Immunogenic composition

ABSTRACT

Immunogenic compositions and methods of their use, as well as processes for their production are provided herein.

FIELD

The disclosure relates to the field of Neisserial immunogeniccompositions and vaccines, their manufacture and the use of suchcompositions in medicine.

BACKGROUND

Neisserial strains of bacteria are the causative agents for a number ofhuman pathologies, against which there is a need for effective vaccinesto be developed. In particular Neisseria gonorrhoeae and Neisseriameningitidis cause pathologies which could be treated by vaccination.

Neisseria meningitidis is an important pathogen, particularly inchildren and young adults. Septicemia and meningitis are the mostlife-threatening forms of invasive meningococcal disease (IMD). Thisdisease has become a worldwide health problem because of its highmorbidity and mortality.

Thirteen N. meningitidis serogroups have been identified based onantigenic differences in the capsular polysaccharides, the most commonbeing A, B and C which are responsible for 90% of disease worldwide.Serogroup B is the most common cause of meningococcal disease in Europe,USA and several countries in Latin America

Vaccines based on the capsular polysaccharide of serogroups A, C, W andY have been developed and have been shown to control outbreaks ofmeningococcal disease (Peltola et al 1985 Pediatrics 76; 91-96). Howeverserogroup B is poorly immunogenic and induces only a transient antibodyresponse of a predominantly IgM isotype (Ala'Aldeen D and Cartwright K1996, J. Infect. 33; 153-157). There is therefore no broadly effectivevaccine currently available against the serogroup B meningococcus whichis responsible for the majority of disease in most temperate countries.This is particularly problematic since the incidence of serotype Bdisease is increasing in Europe, Australia and America, mostly inchildren under 5. The development of a vaccine against serogroup Bmeningococcus presents particular difficulties because thepolysaccharide capsule is poorly immunogenic owing to its immunologicsimilarity to human neural cell adhesion molecule.

Strategies for vaccine production have therefore concentrated on thesurface exposed structures of the meningococcal outer membrane but havebeen hampered by the marked variation in these antigens among strains.

One antigen contemplated for use in vaccines against Neisseriameningitidis is fHbp. Lewis et al discloses the status of fHbp as avaccine candidate Expert Reviews Vaccines 8(6)p729, (2009).

SUMMARY

In a first aspect the present disclosure relates to an immunogeniccomposition comprising:

-   -   (1) a first, fHbp, polypeptide antigen; and    -   (2) a second antigen capable of generating an antibody response        against a Neisseria meningitidis L2 immunotype        for prevention of Neisserial infection or disease.

In a further aspect the present disclosure relates to a method oftreatment or prevention of Neisserial infection or disease comprisingadministering to an individual in need thereof a protective dose of animmunogenic composition comprising:

-   -   (1) a first, fHbp, polypeptide antigen and    -   (2) a second antigen capable of generating an antibody response        against a Neisseria meningitidis L2 immunotype.

In a further aspect the present disclosure relates to an immunogeniccomposition comprising

-   -   (1) a first, fHbp, polypeptide antigen, and    -   (2) a second antigen capable of generating an antibody response        against an Neisserial meningitidis L2 immunotype.

In a further aspect the present disclosure relates to use of

-   -   (1) a first, fHbp, polypeptide antigen and    -   (2) a second antigen capable of generating an antibody response        against an Neisserial meningitidis L2 immunotype        in the preparation of a medicament for prevention of infection        or disease caused by Neisseria infection or disease.

In a further aspect the present disclosure relates to a kit comprising

-   -   (1) a first, fHbp, polypeptide antigen and    -   (2) a second antigen capable of generating an antibody response        against a Neisseria meningitidis L2 immunotype.

In a further aspect the disclosure relates to a method for manufactureof an fHbp based composition for prevention or amelioration of Neisseriameningitidis infection or disease, the method comprising combining anfHbp antigen with a second antigen capable of generating an antibodyresponse against a Neisseria meningitidis L2 immunotype.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Level of expression of fHBP, by Western-Blot. Shows whole-cellsexpressing different level of fHBP: high (line 1), intermediate (line7), low (line 5) and non-detectable (lines 2, 3, 4, 6)

FIG. 2 Alignment of mature fHbp protein sequences. fHbp variant B ofMC58 strain and fHBP variant A of 2996 strain are used as referencesequences. Mature fHBP amino acid sequences were compared with theClustalW method (MegAlign SoftWare from DNASTAR Lasergene 7).

FIG. 3 Tdfl structure

SEQUENCE IDENTIFIERS

The following sequence identifiers are included in the sequence listingand mentioned throughout the description:

SEQ ID NO: 1—amino acid residues 1 to 137 of the mature sequence of afamily B fHbp protein

SEQ ID NO: 2—amino acid residues 136 to 254 of the mature sequence of afamily A fHbp protein

SEQ ID NO: 3—consensus sequence from Family A over region 113-135

SEQ ID NO: 4—consensus sequence from Family B over region 113-135

SEQ ID NO: 5-mature Family B fHbp sequence from strain MC58

SEQ ID NO: 6—nucleic acid sequence for mature Family B fHbp sequencefrom strain MC58

SEQ ID NO: 7—mature Family A fHbp sequence from strain 8047

SEQ ID NO: 8—nucleic acid sequence for mature Family A fHbp sequencefrom strain 8047

SEQ ID NO: 9—amino acid 27 to 273 of full length fHbp sequence fromstrain 8047 with histidine tag (LVL489)

SEQ ID NO: 10—nucleic acid sequence for LVL489

SEQ ID NO: 11—amino acid 73 to 320 of full length fHbp sequence fromstrain MC58 with histidine tag (LVL 490)

SEQ ID NO: 12—nucleic acid sequence for LVL490

SEQ ID NO: 13—amino acid 66-72 of the full length Family B fHbp sequence

SEQ ID NO: 14—sequence of Histidine affinity tag

SEQ ID NO: 15—The peptide GENT (aa residues 136-139 in 8047 of family Aand MC 58 of family B mature sequences) identical in family A and B

SEQ ID NO: 16—amino acid sequence Fusion protein LVL491

SEQ ID NO: 17—nucleic acid sequence for LVL491

SEQ ID NO: 18—amino acid sequence of fusion protein A

SEQ ID NO: 19—nucleic acid sequence for fusion protein A

SEQ ID NO: 20—amino acid sequence of fusion protein B

SEQ ID NO: 21—nucleic acid sequence for fusion protein B

SEQ ID NO: 22—amino acid sequence of fusion protein C

SEQ ID NO: 23—nucleic acid sequence of fusion protein C

SEQ ID NO: 24—amino acid sequence of fusion protein E

SEQ ID NO: 25—nucleic acid sequence of fusion protein E

SEQ ID NO: 26—amino acids within positions 242-246 indicating Family A

SEQ ID NO: 27—amino acids within positions 242-246 indicating Family B

SEQ ID Nos. 28 to 36—mature fHBP protein sequences compared in FIG. 2

DETAILED DESCRIPTION

As discussed elsewhere herein, Neisserial meningitidis L3 immunotypesproduce fHbp at a significantly lower level than strains expressing anL3 inner core LOS. See the comparison studies herein which revealed that6/31 (19%) of L3 strains expressed low or undetectable fHbp, whereas14/15 (93%) of L2 strains expressed low or undetectable fHbp.

Accordingly a vaccine based upon fHbp alone will not be effectiveagainst all menB strains, and fHbp should be supplemented with a secondantigen capable of generating an antibody response against a Neisseriameningitidis L2 immunotype.

The immunogenic compositions of the disclosure are suitably effective asa vaccine against Neisseria meningitides infection or disease.

In a first aspect the present disclosure relates to an immunogeniccomposition comprising an fHbp polypeptide, such as fHbp A or B, or acombination of fHbp polypeptides, or chimeric fHbp. In one aspect thisis a full length fHBP polypeptide or fragment thereof, which is capableof eliciting antibodies against fHbp. Such fHbp may be a fusionpolypeptide as disclosed in, for example, WO/2009/114485. The fHbp maybe from any fHbp family. In one aspect an fHbp polypeptide is anypolypeptide that comprises an epitope capable of eliciting antibodiesagainst fHbp. fHbp is also referred to as as GNA1870 and lipoprotein2086.

In one aspect the fHbp polypeptide is a fusion protein.

The present disclosure identifies 2 families of fHbp protein, A and B,and thus allows for the construction of chimaeric fHbp fusion proteinswith sequences from 2 families, providing protection against Neisseriameningitidis infection or disease.

The terms chimaeric fHbp protein and fHbp fusion protein are usedinterchangeably herein.

In a first aspect, the disclosure relates to a fusion protein comprisingamino acid sequences F1 and F2, where F1 is a N-terminal fragment of afirst fHbp amino acid sequence and F2 is a C-terminal fragment of asecond fHbp amino acid sequence.

In one aspect the fusion protein comprises an F1 N-terminal fragmentfrom fHbp Family B and F2 C-terminal fragment from fHbp Family A.

In one aspect F1 and F2 are at least 10 amino acids in length butsuitably comprise 20 amino acids, more suitably 30 amino acids, moresuitably 40 amino acids, suitably 50 amino acids, suitably 60 aminoacids, suitably 70 amino acids, suitably 80 amino acids, suitably 90amino acids, suitably 100 amino acids, suitably 110 amino acids orsuitably 120 amino acids, suitably taken contiguously from the aminoacid sequence of an fHbp protein.

In one aspect the F1 and F2 fragments do not comprise overlappingsequences, when considered in respect of an alignment between the firstand second fHbp family sequences.

In one aspect the fusion protein F1+F2 has a combined length of at least200 amino acids, suitably at least 210, at least 220, at least 230, atleast 240, at least 250 amino acids, and in one aspect is a full lengthFHBP protein of 254 amino acids.

The 2 parts, F1 and F2, of any fusion protein of the disclosure do notneed to be directly linked, and may include a linker between F1 and F2.However, in one aspect, the F1 and F2 parts are directly linked.

Optionally the fusion protein may have additions, substitutions ordeletions.

In one aspect both the F1 and F2 portions of the sequence comprise animmunogenic epitope.

In one aspect the F1 or F2 portion of the sequence contains epitopessuitable for generation of antibodies against family A and family B fHbpproteins.

In one aspect an N terminal fragment may be equivalent to, or part of,the 140 N-terminal amino acids of the mature sequence of a fHbp family Bprotein, suitably all or part of the N-terminal 135, 136, 137, 138 or139 amino acids of the mature sequence of the fHbp protein.

Amino acid residues 1 to 137 of the mature sequence of a family B fHbpprotein represent amino acid residues 66 to 202 of the full lengthfamily B fHbp sequence.

Suitable fragments are amino acid residues, 1 to 135, 1 to 136, 1 to137, 1 to 138, 1 to 139, 8 to 135, 8 to 137 or 8 to 139 of the maturesequence of a family B fHbp protein. Residues 1 to 137 of the maturesequence are represented by strain MC58 of Family B shown in Seq ID No.1 below or the equivalent regions of other strains of Family B.

SEQ ID No. 1:CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGE

In one aspect, one or more of the first seven amino acids of the maturesequence of the F1 N terminal fragment are replaced by a histidine tag,or other affinity tag, to facilitate purification. In a further aspect ahistidine tag, or other affinity tag, is added to the N terminus of themature protein to facilitate purification.

In one aspect a C-terminal fragment is equivalent to, or part of, aminoacids 130-254 of the mature fHbp sequence of a family A fHbp protein,suitably all or part of amino acids 136-254, 137-254, 138-254, 139-254,or 140 to 254 of the mature sequence.

Amino acid residues 136 to 254 of the mature sequence of a family A fHbpprotein represent amino acid residues 155 to 273 of the full lengthfamily A fHbp sequence.

Some suitable fragments are amino acid residues 136 to 254, 137-254, 138to 254, 139-254, or 140 to 254 of the mature sequence of a family A fHbpprotein. Residues 136 to 254 of the mature sequence are represented bystrain 8047 of Family A shown in Seq ID No. 2 below or the equivalentregions of other strains of Family A.

SEQ ID No. 2:GEHTA FNQLPDGKAE YHGKAFSSDD AGGKLTYTID FAAKQGHGKI EHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVK IGEKVHEIGIAGKQ

Amino acid residues GENT are conserved among family A and B (aa residues136-139 in 8047 of family A and MC 58 of family B) and can therefore beincluded in the amino acid sequence from either family.

FHbp proteins are defined into two families, A and B, herein.

In one aspect the family classification is disclosed in “SequenceDiversity of the Factor H Binding Protein Vaccine Candidate inEpidemiologically Relevant Strains of Serogroup B Neisseriameningitides. The Journal of infectious diseases 2009, vol. 200, no 3,pp. 379-389”

In one aspect the family identity is assessed over region 136-254.

In one aspect proteins in the same family have >80% identity based uponthe sequence of fHbp starting from amino acid 136 of the mature proteinto the C terminus.

In one aspect proteins in different families have 50-75% identity basedupon the sequence of fHbp starting from amino acid 136 of the matureprotein to the C terminus.

In one aspect the family identity is assessed over region 113-135.

In one aspect proteins in the same family have >69% identity based uponthe region 113-135 of the mature amino acid sequence of fHbp.

In one aspect proteins in different families have <20% identity basedupon the region 113-135 of the mature amino acid sequence of fHbp.

In one aspect Family A and B may be distinguished by the presence of oneor more of the following amino acids:

AA position Family A Family B  98 I V 102 D/N S 106-107 VV LT 111 I T140 A S 142-143 NQ D/GK 146 No amino acid equivalent to E/K position 146in family B. 149 K m/r/s 151 E T 153 H R 155 K T 158 S G 186 T S 197-198EL D/YI 200 A P 204 S R/H 209 L S 211-213 DTR SVL 215-217 GG/SE NQA/D221 T S 223 H S 225-226 AL GI 229-230 DR GK/Q 234 I V 239 T E 242-246IG/REKV (SEQ ID NO. 26) TA/VNGI (SEQ ID No. 27) 248 E H 251 I L 253 G A

In one aspect family A and B comprises the following consensus sequencefrom region 113-135:

(SEQ ID NO. 3) A KINNPDK(I/T)DSLIN(Q/R)RSFLVSGLG (SEQ ID NO. 4) BQ(V/I/E)QD(S/P)E(D/H)S(G/R)(K/S)MVAKR(Q/R)F(R/K)IGDI(A/V)

An example of a family B sequence (SEQ ID NO. 5) is strain MC58:

  1 CSSGGGGVAA DIGAGLADAL TAPLDHKDKG LQSLTLDQSV  RKNEKLKLAA  51QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QIEVDGQLIT  LESGEFQVYK 101QSHSALTAFQ TEQIQDSEHS GKMVAKRQFR IGDIAGEHTS  FDKLPEGGR A 151TYRGTAFGSD DAGGKLTYTI DFAAKQGNGK IEHLKSPELN                   *                      * VDLAAADIKP 201 DGK RHAVISG SVLYNQAEKG SYSLGIFGGK AQEVAGSAEV  KTVNGIRHIG 251 LAAKQ

Amino acids identified within this sequence as being of potentialimportance include:

-   Gly121 and Lys122: residues essential for the binding of MAbs JAR3    and 5-   Peptide Glu146→Arg149 and Arg204: residues essential for the binding    of MAb502-   Residues Pro145, Phe227, Gly228, Lys230 and Glu233: could    potentially play a minor role in MAb502 recognition-   Glu218 and Glu239 (*):involved in factor H-binding

Corresponding nucleic sequence (SEQ ID No. 6):

  1 TGCAGCAGCG GAGGGGGTGG TGTCGCCGCC GACATCGGTG CGGGGCTTGC  51CGATGCACTA ACCGCACCGC TCGACCATAA AGACAAAGGT TTGCAGTCTT 101TGACGCTGGA TCAGTCCGTC AGGAAAAACG AGAAACTGAA GCTGGCGGCA 151CAAGGTGCGG AAAAAACTTA TGGAAACGGT GACAGCCTCA ATACGGGCAA 201ATTGAAGAAC GACAAGGTCA GCCGTTTCGA CTTTATCCGC CAAATCGAAG 251TGGACGGGCA GCTCATTACC TTGGAGAGTG GAGAGTTCCA AGTATACAAA 301CAAAGCCATT CCGCCTTAAC CGCCTTTCAG ACCGAGCAAA TACAAGATTC 351GGAGCATTCC GGGAAGATGG TTGCGAAACG CCAGTTCAGA ATCGGCGACA 401TAGCGGGCGA ACATACATCT TTTGACAAGC TTCCCGAAGG CGGCAGGGCG 451ACATATCGCG GGACGGCGTT CGGTTCAGAC GATGCCGGCG GAAAACTGAC 501CTACACCATA GATTTCGCCG CCAAGCAGGG AAACGGCAAA ATCGAACATT 551TGAAATCGCC AGAACTCAAT GTCGACCTGG CCGCCGCCGA TATCAAGCCG 601GATGGAAAAC GCCATGCCGT CATCAGCGGT TCCGTCCTTT ACAACCAAGC 651CGAGAAAGGC AGTTACTCCC TCGGTATCTT TGGCGGAAAA GCCCAGGAAG 701TTGCCGGCAG CGCGGAAGTG AAAACCGTAA ACGGCATACG CCATATCGGC 751CTTGCCGCCA AGCAATAA

Other examples of family B species include strains H44/76, M982,M060240006, 03s-0408, and other examples will be well known to theskilled person.

An example of a family A sequence (SEQ ID NO. 7) is strain 8047:

  1 CSSGGGGVAA DIGARLADAL TAPLDHKDKS LQSLTLDQSV RKNEKLKLAA  51QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QIEVDGQLIT LESGEFQIYK 101QDHSAVVALQ IEKINNPDKI DSLINQRSFL VSGLGGEHTA FNQLPDGKAE 151YHGKAFSSDD AGGKLTYTID FAAKQGHGKI EHLKTPEQNV                 *                      * ELAAAELKAD 201 EKSHAVILGD TRYGS EEKGT YHLALFGDRA QEIAGSATVK IGEKVHEIGI 251 AGKQ

Amino acids identified within this sequence as being of potentialimportance include:

-   Ala173: residue essential for the binding of MAb JAR11-   Lys179 and Glu191: residues essential for the binding of MAb JAR10-   Ser215: residue essential for the binding of MAb JAR13-   Glu217 (*) and Glu238: involved in factor H-binding. Remark: the    second glutamate could be replaced by a Thr238 (*) in some strains    (it is the case in the strain 8047). These strains can also bind the    factor H.

Corresponding nucleic sequence (SEQ ID NO. 8):

  1 TGCAGCAGCG GAGGCGGCGG TGTCGCCGCC GACATCGGCG CGAGGCTTGC  51CGATGCACTA ACCGCACCGC TCGACCATAA AGACAAAAGT TTGCAGTCTT 101TGACGCTGGA TCAGTCCGTC AGGAAAAACG AGAAACTGAA GCTGGCGGCA 151CAAGGTGCGG AAAAAACTTA TGGAAACGGC GACAGCCTCA ATACGGGCAA 201ATTGAAGAAC GACAAGGTCA GCCGCTTCGA CTTTATCCGT CAAATCGAAG 251TGGACGGGCA GCTCATTACC TTGGAGAGCG GAGAGTTCCA AATATACAAA 301CAGGACCACT CCGCCGTCGT TGCCCTACAG ATTGAAAAAA TCAACAACCC 351CGACAAAATC GACAGCCTGA TAAACCAACG CTCCTTCCTT GTCAGCGGTT 401TGGGCGGAGA ACATACCGCC TTCAACCAAC TGCCTGACGG CAAAGCCGAG 451TATCACGGCA AAGCATTCAG CTCCGACGAT GCTGGCGGAA AACTGACCTA 501TACCATAGAT TTCGCCGCCA AACAGGGACA CGGCAAAATC GAACACCTGA 551AAACACCCGA GCAAAATGTC GAGCTTGCCG CCGCCGAACT CAAAGCAGAT 601GAAAAATCAC ACGCCGTCAT TTTGGGCGAC ACGCGCTACG GCAGCGAAGA 651AAAAGGCACT TACCACCTCG CCCTTTTCGG CGACCGCGCC CAAGAAATCG 701CCGGCTCGGC AACCGTGAAG ATAGGGGAAA AGGTTCACGA AATCGGCATC 751GCCGGCAAAC AGTAG

Other examples of family A species include strains M1239, M981,M08_(—)240117, M97252153, and other examples will be well known to theskilled person.

The fusion protein is suitably capable of eliciting antibodies againstboth family members A and B as defined herein. In one aspect the fusionprotein is able to elicit neutralizing antibodies, suitably in responseto infection by N. meningitidis expressing family A or family B fHbpmolecules.

Suitably the chimaeric protein, when administered at an effective dose,elicits a protective immune response against Neisserial infection, moresuitably protective against N. meningitidis serogroup B infection.

In one aspect the fusion protein is immunologically reactive withantibodies generated against Neisserial full-length fHbp proteins orwith antibodies generated by infection of a mammalian host withNeisseria.

In one aspect chimeric proteins are able to elicit the production ofbactericidal antibodies mediating the complement killing of strainsexpressing either the fHbp A or fHbp B.

In one aspect the fusion protein of the disclosure has at least one atleast one mutation to prevent or reduce Factor H binding.

In one aspect the mutation is a deletion, insertion or substitution.Factor H binding may be human factor H binding, for example as assessedby ELISA or surface plasmon resonance, as disclosed in M. C. Schneideret al., 2009 “Neisseria meningitidis recruits factor H using proteinmimicry of host carbohydrates” Nature Letters.

In one aspect the mutation to prevent factor H binding is containedwithin the C-terminal F2 fragment, from amino acids 136-254 of fHbp.

In one aspect the mutation to prevent factor H binding comprisessubstituting at least one Glu to Ala in fHbp. In a further aspect themutation to prevent factor H binding comprises substituting one or moreof the following residues contained within an F2 fragment to preventfHbp binding: Glu 217, Glu/Thr 238 of fHbp Family A; Glu 218, Glu 239 offHbp Family B. In one aspect one or more of the residues are mutated toalanine.

Factor H binding may be assessed by ELISA. For example, fHbppoylpeptides (chimeric or not) may be coated on a microplate. Aftersaturation and washes, purified human fH or recombinant human fH isincubated in microwells and binding to fHBP is revealed (after washes)via addition of rabbit antibodies directed against the human fH andsubsequent incubation with anti-rabbit IgG conjugated to peroxidise.

In one aspect, where the F1 fragment is from family B, the F1 fragmentcomprises both Gly at position 121 and Lys at position 122, (numberingbased on the MC58 strain as a reference family B strain).

In one aspect the fusion protein is capable of binding to antibodiesJAR3 or JAR5

In one aspect, where the F2 fragment is from family A, the F2 fragmentcomprises one, or more or all of the following amino acids (numberingbased on the 8047 strain as a reference A strain): Ala 173; Ser 215; Lys179 and Glu 191, and in one aspect comprises both Lys 179 and Glu 191.In one aspect the fusion protein is capable of binding to one or more ofantibodies JAR10, JAR11, JAR13.

In one aspect, where the F2 fragment is from family A, the fusionprotein being constructed to comprise one or more or all of thefollowing amino acids replacing the naturally occurring amino acids:ala217, optionally ala at position 238, Glu146, Gly inserted at position146, after the glutamine (subsequent numbers being shifted by +1 withrespect to the wild type 8047 sequence), Gly148, Arg149 and Arg204(numbering based on the MC58 strain as a reference family B strain). Inone aspect the fusion protein is capable of binding to MAb502

In one aspect, where F2 fragment is from family A, then the fragment maycomprises one or more or all of pro 145, phe 227, gly 228, lys 230 andglu 233. In one aspect the fusion protein is capable of binding toMAb502.

Antibodies mentioned above are referred to in the followingpublications, herein fully incorporated by reference: P. T. Beernink andD. M. Granoff, 2008 “Bactericidal antibody induced by meningococcalsrecombinant chimeric factor H-binding protein vaccines” Inf. & Imm. vol.76, p. 2568-2575; P. T. Beernink et al., 2008 “Fine antigenicspecificity and cooperative bactericidal activity of monoclonalantibodies directed at the meningococcal vaccine candidate FactorH-binding protein” Inf. & Imm. vol. 76, p. 4232-4240; M. Scarselli etal., 2009 “Epitope mapping of a bactericidal monoclonal antibody againstthe factor H binding protein of Neisseria meningitidis” J. Mol. Biol.vol. 386, p. 97-108). In one aspect fHbp includes amino acids disclosedas being relevant for immunogenicity in any such publication.

In one aspect the chimaeric protein of the disclosure comprising one ormore amino acid alterations as defined above, demonstrates an increaseof the bactericidal titers against strains expressing fHbp compared tothe chimeric proteins without these modifications.

In one aspect the fusion protein of the present disclosure comprisesresidues 1 to 135, 1 to 136, 1 to 137, 1 to 138 or 1 to 139 from amature family B fHbp protein and residues 136 to 254, 137 to 254, 138 to254, 139 to 254 or 140 to 254 of a mature family A fHbp protein, and isunable to bind to Factor H.

In one aspect the fusion protein of the present disclosure comprisesresidues 8 to 135, 8 to 136, 8 to 137, 8 to 138 or 8 to 139 from amature family B fHbp protein, optionally with a histidine tag, andresidues 136 to 254, 137 to 254, 138 to 254, 139 to 254 or 140 to 254 ofa mature family A fHbp protein, and is unable to bind to Factor H.

In one aspect, one or more of the first seven amino acids from a maturefamily B fHbp protein are absent and may be replaced by a histidine tag,or other affinity tag, to facilitate purification. In such cases, thefamily B fHbp portion of the fusion protein starts at residue 2, 3, 4,5, 6 or 7 of the mature sequence.

In one aspect the fusion polypeptide comprises residues 1 to 135, 1 to136, 1 to 137, 1 to 138, or 1 to 139 from a family B fHbp protein, forexample having the MC58 sequence, and residues 136 to 254, 137 to 254,138 to 254, 139 to 254 or 140 to 254 of a family A fHbp protein, forexample having the sequence of strain 8047, the fusion protein beingconstructed to comprise the following amino acids replacing thenaturally occurring amino acids: Ala217, optionally Ala at position 238,Glu146, Gly inserted at position 146, after the glutamine (subsequentnumbers being shifted by +1 with respect to the wild type 8047sequence), Gly148, Arg149 and Arg204 of family B mature protein sequencefrom strain MC58. These amino acids are found in construct C exemplifiedbelow. In one aspect the fusion polypeptide additionally comprises oneor more or all of pro 145, phe 227, gly 228, lys 230 and glu 233. Theseamino acids are found in construct E exemplified below.

In one aspect the fusion polypeptide is selected from fusion proteinsLVL491 (SEQ ID NO. 16), A (SEQ ID NO. 18), B (SEQ ID NO. 20), C (SEQ IDNO. 22), D, E (SEQ ID NO. 24) or F as disclosed herein, particularlyfrom fusion proteins A, B, C, and E.

The composition also comprises a second antigen capable of generating anantibody response against a Neisseria meningitidis L2 immunotype.

Reference or claim to any specific antigen herein includes all deletion,insertion and substitution mutations of that antigen, or other specificvariant of that antigen as described herein, or (where the antigen is apolypeptide) to polypeptides having 80% or more, suitably at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% identity to that polypeptide, suitably being immunogenic.

Reference to an immunogenic composition comprising both antigens hereinis intended to encompass true combinations of different antigens forcombined delivery, for example in the form of a single vaccine dose, aswell as an immunogenic composition comprising both antigens forsimultaneous delivery, or substantially simultaneous delivery (forexample by an injection of each component on the same visit to a medicalpractitioner), as well as a sequential delivery of one antigen followed,after a time interval, with delivery of a second antigen. Thus theimmunogenic composition of the disclosure may be unitary or compriseseparable components for combined or sequential delivery, asappropriate.

The antigen capable of generating an antibody response against aNeisseria meningitidis L2 immunotype may be any suitable antigen capableof generating an immune response which protects or ameliorates theinfection or disease caused by infection by >50%, >60%, >70%, >80%or >90% of L2 immunotypes, suitably an antigen capable of generating animmune response which protects against or ameliorates the infection ordisease caused by infection by the L2 immunotype.

In one aspect the antigen is one which is encoded or expressedby >50%, >60%, >70%, >80% or >90% of Neisseria meningitidis L2immunotypes, more suitably substantially all of Neisseria meningitidisL2 immunotypes, and more suitably wherein the % expression is determinedin respect of the strains circulating in a given country or region.

In one aspect the antigen is selected from L2 LOS, Tdfl, Hsf or Hap, oris selected from the group consisting of a combination of two or more ofsaid antigens such as L2 LOS and Tdfl, L2 LOS and Hap, L2 LOS and Hsf,Tdfl and Hap, Tdfl and Hsf, Hap and Hsf, Tdfl and Hap and Hsf, L2 LOSand Tdfl and Hap, L2 LOS and Tdfl and Hsf, and L2 LOS and Hap and Hsf.These antigens are discussed in more detail below.

Hsf: Hsf has a structure that is common to autotransporter proteins. Forexample, Hsf from N. meningitides strain H44/76 consists of a signalsequence made up of amino acids 1-51, a head region at the aminoterminus of the mature protein (amino acids 52-479) that is surfaceexposed and contains variable regions (amino acids 52-106, 121-124,191-210 and 230-234), a neck region (amino acids 480-509), a hydrophobicalpha-helix region (amino acids 518-529) and an anchoring domain inwhich four transmembrane strands span the outer membrane (amino acids539-591).

Although full length Hsf may be used in immunogenic compositions of thedisclosure, various Hsf truncates and deletions may also be useddepending on the type of immunogenic composition or vaccine.

Where Hsf is used in a subunit composition or vaccine, a portion of thesoluble passenger domain may be used; for instance the complete domainof amino acids 52 to 479, most suitably a conserved portion thereof, forinstance the sequence of amino acids 134 to 479. Suitable forms of Hsfmay be truncated so as to delete variable regions of the proteindisclosed in WO01/55182.

Suitable variants would include the deletion of one, two, three, four,or five variable regions as defined in WO01/55182. The above sequencesand those described below, can be extended or truncated by up to 1, 3,5, 7, 10 or 15 amino acids at either or both N or C termini.

Suitable fragments of Hsf therefore include the entire head region ofHsf, suitably containing amino acids 52-473. Additional suitablefragments of Hsf include surface exposed regions of the head includingone or more of the following amino acid sequences; 52-62, 76-93,116-134, 147-157, 157-175, 199-211, 230-252, 252-270, 284-306, 328-338,362-391, 408-418, 430-440 and 469-479.

Where Hsf is present in an outer membrane vesicle preparation, it may beexpressed as the full-length protein or as a variant made up of a fusionof amino acids 1-51 and 134-591 (yielding a mature outer membraneprotein of amino acid sequence 134 to the C-terminus). Suitable forms ofHsf may be truncated so as to delete variable regions of the proteindisclosed in WO01/55182. Suitable variants would include the deletion ofone, two, three, four, or five variable regions as defined inWO01/55182. In one aspect the first and second variable regions aredeleted.

Suitable variants would delete residues from between amino acid sequence52 through to 237 or 54 through to 237, more suitably deleting residuesbetween amino acid 52 through to 133 or 55 through to 133. The matureprotein would lack the signal peptide.

Hap: Computer analysis of the Hap-like protein from Neisseriameningitidis reveals at least three structural domains. Considering theHap-like sequence from strain H44/76 as a reference, Domain 1,comprising amino-acid 1 to 42, encodes a sec-dependant signal peptidecharacteristic of the auto-transporter family, Domain 2, comprisingamino-acids 43 to 950, encode the passenger domain likely to be surfaceexposed and accessible to the immune system, Domain 3, comprisingresidues 951 to the C-terminus (1457), is predicted to encode abeta-strands likely to assemble into a barrel-like structure and to beanchored into the outer-membrane. Since domains 2 is likely to besurface-exposed, well conserved (more than 80% in all strain tested) andcould be produced as subunit antigens in E. coli, it represents aninteresting vaccine candidate. Since domains 2 and 3 are likely to besurface-exposed, are well conserved (Pizza et al. (2000), Science 287:1816-1820), they represent interesting vaccine candidates. Domain 2 isknown as the passenger domain.

Immunogenic compositions of the disclosure may comprise the full-lengthHap protein, suitably incorporated into an OMV preparation. Immunogeniccompositions of the disclosure may also comprise the passenger domain ofHap which in strain H44/76 is composed of amino acid residues 43-950, orthe N-terminal fragment from residues 43-1178. These fragments of Hapwould be particularly advantageously used in a subunit composition ofthe disclosure. The above sequence for the passenger domain of Hap (orN-terminal fragment) can be extended or truncated by up to 1, 3, 5, 7,10, 15, 20, 25, or 30 amino acids at either or both N or C termini.

Tdfl: Neisserial antigen NMB0964 (NMB numbers refer to Neisseriameningitidis group B genome sequences available from www.neisseria.org)[known as NMA1161 in the Neisseria meningitidis group A genome of strainZ2491, and as BASB082 in WO 00/55327, and as ZnuD] is a conservedantigen throughout Neisseria and can induce bactericidal antibodiesagainst a range of neisserial strains. The inventors have found thisantigen functions as a Zn²⁺ receptor in the bacterium, and itsexpression is regulated by the level of Zn²⁺ in the medium.

By the term NMB0964 polypeptide herein it includes the neisserial Tdflpolypeptide (encoded by the tdfl gene) in general from any neisserialstrain (the protein is so well conserved amongst neisserial strains itsidentity in any particular neisserial strain is readily ascertainable bypersons skilled in the art). The term therefore includes the NMA1161sequence, and the BASB082 polypeptide sequence (and all the Polypeptidesof the Disclosure concerning the BASB082 polypeptide) of WO 00/55327.For instance the NMB0964 polypeptide of the disclosure will cover SEQ IDNO: 2 of WO00/55327 or polypeptides with more than 70, 80, 90 or 95%sequence identity with said SEQ ID NO:2, or polypeptides comprisingimmunogenic fragments of 7, 10, 12, 15 or 20 (or more) contiguous aminoacids from said SEQ ID NO: 2 (in particular said immunogenic fragmentsbeing capable of eliciting—if necessary when coupled to a proteincarrier—an immune response which can recognise said SEQ ID NO: 2).Particularly suitable NMB0964 immunogenic fragment embodiments are thoseextracellular loop sequences shown in the topology diagram of FIG. 3 asapplied to any given NMB0964 sequence. In particular the thirdextracellular loop is provided (wherein the 2 Cys residues areoptionally disulphide linked or not). Said NMB0964 immunogenic fragmentpolypeptide sequences may have more than 70, 80, 90 or 95% sequenceidentity with said extracellular loop sequences (as defined in FIG. 3)from SEQ ID NO:2 of WO 00/55327, or may be polypeptides comprisingimmunogenic fragments of 7, 10, 12, 15 or 20 (or more) contiguous aminoacids from said extracellular loop sequences (as defined in FIG. 3) fromSEQ ID NO: 2 (in particular said immunogenic fragments being capable ofeliciting—if necessary when coupled to a protein carrier—an immuneresponse which can recognise said SEQ ID NO: 2) and are provided asNMB0964 polypeptides of the disclosure. Said NMB0964 immunogenicfragment polypeptide sequences may have more than 70, 80, 90, 95, 99 or100% sequence identity with the sequence from the third extracellularloop sequence given in FIG. 3 (wherein optionally the 2 Cys residuesshould be conserved, and may or may not be disulphide linked), or may bepolypeptides comprising immunogenic fragments of 7, 10, 12, 15 or 20 (ormore) contiguous amino acids from said extracellular loop sequence (inparticular said immunogenic fragments being capable of eliciting—ifnecessary when coupled to a protein carrier—an immune response which canrecognise SEQ ID NO: 2 of WO00/55327) and are provided as NMB0964polypeptides of the disclosure. In one embodiment the NMB0964immunogenic fragment polypeptides are not full-length NMB0964 (maturesequence or with signal sequence) polypeptides.

In one aspect NMB0964 may be used as an isolated antigen in a subunitcomposition or vaccine approach.

In another aspect the NMB0964 antigen may be used in the form ofisolated outer membrane vesicles prepared from a Neisseria speciesbacterium, wherein the Neisseria species bacterium produces a level of aNMB0964 polypeptide sufficient to provide for production of a vesiclethat, when administered to a subject, elicits anti-NMB0964 antibodies;and a pharmaceutically acceptable excipient.

This may be achieved due to the Neisseria species bacterium beinggenetically modified in NMB0964 polypeptide production by for instance:disrupting the functional expression of the Zur repressor (NMB1266)—aprotein which switches off expression of NMB0964 in the presence of Zn²⁺in the medium; replacing the NMB0964 promoter with one that does notbind Zur, in particular with a stronger promoter than the endogenousNMB0964 promoter such as a lac promoter; or through using a medium lowin Zn²⁺ concentration—i.e. under 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5,0.4, 0.3, 0.2, 0.1, 0.05 or 0.01 μM free Zn²⁺—(such as Roswell ParkMemorial Institute medium 1640 (RPMI) which has around 1.69 μM Zn²⁺ byICP-MS), or removing Zn²⁺ in the medium, for instance using a known zincchelator such as TPEN(N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine)—enough should beadded to the medium such that the expression of the NMB0964 ismaximised.

The Neisseria species bacterium may be deficient in capsularpolysaccharide, for instance through disruption of functional expressionof the siaD gene. It may be disrupted in the functional expression ofthe msbB and/or htrB genes to detoxify the LOS in the outer membranevesicle. It may be disrupted in the expression of one or more thefollowing genes: PorA, PorB, OpA, OpC, PilC, or FrpB. It may bedisrupted in the functional expression of the IgtB gene. Such disruptionmethods are described in WO 01/09350 and WO2004/014417. The Neisseriaspecies bacterium may be of immunotype L2 or L3.

Methods for the preparation or isolation of outer membrane vesicles(also known as microvesicles or blebs) from Neisserial strains are wellknown in the art, and are described in WO 01/09350 and WO2004/014417,and also below. Typically outer membrane vesicles are isolated byextracting either without a detergent, or with 0-0.5, 0.02-0.4,0.04-0.3, 0.06-0.2, or 0.08-0.15% detergent, for instance deoxycholate,e.g. with around or exactly 0.1% deoxycholate.

An OMV composition or vaccine prepared either in specific cultureconditions low in Zn2+, or from a mutant N. meningitidis strainengineered to either over-express NMB0964 or to remove the Zincrepression mechanism mediated through Zur, is enriched in NMB0964, andsuch OMVs may elicit good bactericidal antibody responses compared toOMVs which have not been prepared with these methods.

In one aspect the disclosure relates to an immunogenic compositioncomprising an isolated outer membrane vesicles prepared from a Neisseriaspecies bacterium, wherein the Neisseria species bacterium produces alevel of a NMB0964 polypeptide sufficient to provide for production of avesicle that, when administered to a subject, elicits anti-NMB0964antibodies; and a pharmaceutically acceptable excipient. The NMB0964polypeptide may be endogenous to the Neisseria species bacterium. TheNeisseria species bacterium may be genetically modified to contain anucleic acid encoding an exogenous

NMB0964 polypeptide. The NMB0964 polypeptide may be expressed from anNMB0964 gene with an endogenous promoter. The Neisseria speciesbacterium may be genetically modified in NMB0964 polypeptide production.The Neisseria species bacterium may be genetically modified through thedisruption of functional expression of the Zur repressor (NMB1266).

The Neisseria species bacterium may be genetically modified to providefor expression of a NMB0964 polypeptide from a heterologous promoter.The heterologous promoter in one aspect does not bind the Zur repressor.In one aspect the heterologous promoter is a stronger promoter in theNeisserial species bacterium than the non-repressed endogenous promoterof the NMB0964 gene. In one aspect the heterologous promoter is anIPTG-inducible lac promoter.

In one aspect the level of NMB0964 polypeptide produced by the Neisseriaspecies bacterium is greater than that made by N. meningitidis strainH44/76 grown in tryptic soy broth (TSB). In one aspect level of NMB0964polypeptide produced by the Neisseria species bacterium is the same orgreater than that made by N. meningitidis strain H44/76 grown in RoswellPark Memorial Institute medium 1640 (RPMI). In one aspect the level ofNMB0964 polypeptide produced by the Neisseria species bacterium is thesame or greater than that made by N. meningitidis strain H44/76 grown inRoswell Park Memorial Institute medium 1640 (RPMI) with 1 μM TPEN(N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine). In one aspect thelevel of NMB0964 polypeptide produced by the Neisseria species bacteriumis the same or greater than that made by N. meningitidis strain H44/76in a medium which has less than 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5,0.4, 0.3, 0.2, 0.1, 0.05 or 0.01 μM free Zn²⁺.

In one aspect the Neisserial species bacterium is Neisseriameningitidis, or Neisseria meningitidis serogroup B. In one aspect theNeisseria species bacterium is deficient in capsular polysaccharide. Inone aspect the Neisseria species bacterium is deficient in capsularpolysaccharide through disruption of functional expression of the siaDgene. In one aspect the Neisseria species bacterium is disrupted in thefunctional expression of the msbB and/or htrB genes. In one aspect theNeisseria species bacterium is disrupted in the expression of one ormore the following genes: PorA, PorB, OpA, OpC, PilC, or FrpB. In oneaspect the Neisseria species bacterium is disrupted in the functionalexpression of the IgtB gene. In one aspect wherein the Neisseria speciesbacterium is of immunotype L2 or L3.

In one aspect the outer membrane vesicles are isolated by extractingwith 0-0.5, 0.02-0.4, 0.04-0.3, 0.06-0.2, or 0.08-0.15% detergent, forinstance deoxycholate, e.g. with around or exactly 0.1% deoxycholate.

In one aspect the disclosure relates to a method of producing animmunogenic composition, the method comprising: culturing a Neisseriaspecies bacterium producing a NMB0964 polypeptide, wherein the NMB0964polypeptide is produced at a level sufficient to provide for productionof outer membrane vesicles that, when administered to a subject, elicitanti-NMB0964 antibodies; preparing outer membrane vesicles from thecultured bacterium; and combining the outer membrane vesicles with apharmaceutically acceptable excipient to produce an immunogeniccomposition suitable for administration to a subject in combination withan fHbp polypeptide. In one aspect the NMB0964 polypeptide is endogenousto the Neisseria species bacterium. In one aspect wherein the Neisseriaspecies bacterium has been genetically modified to contain a nucleicacid encoding an exogenous NMB0964 polypeptide. In one aspect theNMB0964 polypeptide is expressed from an NMB0964 gene with an endogenouspromoter. In one aspect the Neisseria species bacterium has beengenetically modified in NMB0964 polypeptide production. In one aspectthe Neisserial species bacterium has been genetically modified throughthe disruption of functional expression of the Zur repressor (NMB1266).In one aspect wherein the Neisseria species bacterium has beengenetically modified to provide for expression of a NMB0964 polypeptidefrom a heterologous promoter. In one aspect the heterologous promoterdoes not bind the Zur repressor. In one aspect heterologous promoter isa stronger promoter in the Neisserial species bacterium than thenon-repressed endogenous promoter of the NMB0964 gene. In one aspect theheterologous promoter is an IPTG-inducible lac promoter. In one aspectthe level of NMB0964 polypeptide produced by the Neisseria speciesbacterium is greater than that made by N. meningitidis strain H44/76grown in tryptic soy broth (TSB). In one aspect the level of NMB0964polypeptide produced by the Neisseria species bacterium is the same orgreater than that made by N. meningitidis strain H44/76 grown in RoswellPark Memorial Institute medium 1640 (RPMI). In one aspect the level ofNMB0964 polypeptide produced by the Neisseria species bacterium is thesame or greater than that made by N. meningitidis strain H44/76 grown inRoswell Park Memorial Institute medium 1640 (RPMI) with 1 μM TPEN(N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine). In one aspect thelevel of NMB0964 polypeptide produced by the Neisseria species bacteriumis the same or greater than that made by N. meningitidis strain H44/76in a medium which has less than 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5,0.4, 0.3, 0.2, 0.1, 0.05 or 0.01 μM free Zn²⁺. In one aspect culturingof the Neisseria species bacterium is in a medium which has less than 5,4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05 or 0.01 μMfree Zn²⁺.

In one aspect culturing of the Neisserial species bacterium is in amedium comprising a Zn²⁺ chelator. In one aspect Zn²⁺ chelator ispresent in the medium at a concentration of 0.01-100, 0.1-10, 0.3-5, or0.5-1 μM. In one aspect the Zn²⁺ chelator present in the medium is TPEN,suitably at a concentration in the range of 1 to 25 μM, suitably aconcentration of 5 μM, 10 μM, 15 μM, 20 μM or 25 μM.

In one aspect the step of preparing outer membrane vesicles is carriedout by extracting with 0-0.5, 0.02-0.4, 0.04-0.3, 0.06-0.2, or0.08-0.15% detergent, for instance deoxycholate, e.g. with around orexactly 0.1-0.5% deoxycholate. In one aspect the step of preparing outermembrane vesicles is carried out without use of a detergent.

In one aspect the fHbp polypeptide is combined with a peptide sequencesharing more than 50, 60, 70, 80, 90, 95, 99, or of 100% sequenceidentity with the following sequence:RDQYGLPAHSHEYDDCHADIIWQKSLINKRYLQLYPHLLTEEDIDYDNPGLSCGFHDDDNA HAHTHS, ora polypeptide comprising an immunogenic fragment of 7, 10, 12, 15 or 20(or more) contiguous amino acids from said sequence (optionally whereinsaid peptide sequence or said immunogenic fragment is capable ofeliciting—if necessary when coupled to a protein carrier—an immuneresponse which can recognise SEQ ID NO: 2 of WO00/55327), and apharmaceutically acceptable carrier. In one aspect the two Cys residuesare present in the polypeptide, and may di-sulphide linked. In oneaspect the polypeptide is not a full length mature NMB0964 polypeptide,or is not a full length NMB0964 polypeptide with signal sequence intact.

The L2 Lipooligosaccharide (L2 LOS)

LPS (lipopolysaccharide, also known as LOS-lipooligosaccharide) is theendotoxin on the outer membrane of Neisseria. The polysaccharide moietyof the LPS is known to induce bactericidal antibodies. The structure andfunction of Los is described inn Verheul Microbiological reviews, March1993, Vol 57, no 1, p34-49.

Heterogeneity within the oligosaccharide moiety of the LPS generatesstructural and antigenic diversity among different neisserial strains(Griffiss et al. Inf. Immun. 1987; 55: 1792-1800). This has been used tosubdivide meningococcal strains into L2 immunotypes (Scholtan et al. JMed Microbiol 1994, 41: 236-243). Immunotypes L3, L7, & L9 areimmunologically and structurally similar (or even the same) and havetherefore been designated L3, 7, 9 (or, for the purposes of thisspecification, generically as“L3”). Meningococcal LPS L3, 7, 9 (L3), L2and L5 can be modified by sialylation, or by the addition of cytidine5′-monophosphate-N-acetylneuraminic acid. See M. P. Jennings et al,Microbiology 1999, 145, 3013-3021 and Mol Microbiol 2002, 43: 931-43 forfurther illustration of LPS structure and heterogeneity.

L2 LOS defines the L2 immunotype and thus is a potential antigen tosupplement an fHbp based composition or vaccine.

L2 LOS suitably generates antibodies capable of killing a Neisserial L2immunotype. In one aspect reference to L2 LOS herein includes L3Vstrains.

L2 LOS may be presented in an outer membrane vesicle (OMV—the term‘bleb’ and ‘outer membrane vesicle’ are used interchangeably herein),suitably where the vesicle is extracted with a low percentage detergent,more suitably 0-0.5%, 0.02-0.4%, 0.04-0.3%, 0.06-0.2%, 0.08-0.15% or0.1%, most suitably deoxycholate [DOC]) but may also be part of asubunit composition or vaccine.

More generally, OMVs of the present disclosure may be extracted using alow percentage detergent, more suitably 0-0.5%, 0.02-0.4%, 0.04-0.3%,0.06-0.2%, 0.08-0.15% or 0.1%, most suitably deoxycholate [DOC])

LOS may be used plain or conjugated to a source of T-cell epitopes suchas tetanus toxoid, Diphtheria toxoid, CRM-197 or OMV outer membraneproteins.

L2 LOS may be detoxified.

Details of all such aspects are described in more detail below, inrelation to the choice and preparation of LOS.

Where LPS, suitably meningococcal LPS, is included in a composition orvaccine of the disclosure, suitably either or both of immunotypes L2 andL3 are present.

In one aspect the second antigen is a meningococcal LPS having a least aPEA on position 6 on Hep II of inner core with or without a second PEAor a glucose on this Hep II.

The combination of the disclosure may be used with other antigens forcombined, simultaneous or sequential delivery.

In one aspect of the disclosure the combination of the disclosureincludes an antigen that is also effective against ST269, whichreference includes the ST269 clonal complex. In one aspect the antigeneffective against ST269 clonal complex is selected from Hap, Tdfl andHsf (discussed in more detail below), or is selected from the groupconsisting of a combination of two or more of said antigens such as Tdfland Hap, Tdfl and Hsf, Hap and Hsf, and Tdfl and Hap and Hsf.

In one aspect of the disclosure the combination of the disclosureincludes an antigen that is also effective against ST11, which referenceincludes the ST11 clonal complex. In one aspect the antigen effectiveagainst ST11 clonal complex is selected from L2 LOS, Hap, Tdfl and Hsf(discussed in more detail below), or is selected from the groupconsisting of a combination of two or more of said antigens such as L2LOS and Tdfl, L2 LOS and Hap, L2 LOS and Hsf, Tdfl and Hap, Tdfl andHsf, Hap and Hsf, Tdfl and Hap and Hsf, L2 LOS and Tdfl and Hap, L2 LOSand Tdfl and Hsf, and L2 LOS and Hap and Hsf.

Sequence types are identified by Multilocus sequence typing (MLST),which characterises isolates of bacterial species using the sequences ofinternal fragments of seven house-keeping genes. For each house-keepinggene, the different sequences present within a bacterial species areassigned as distinct alleles and, for each isolate, the alleles at eachof the seven loci define the allelic profile or sequence type (ST).Sequence types are grouped into clonal complexes by their similarity toa central allelic profile (genotype). For Neisseria, generally once acentral genotype has been identified, clonal complexes are defined asincluding any ST that matches the central genotype at four or more lociunless it more closely matches another central genotype. Reference toNeisserial meningitidis ST269 or ST11 herein includes reference toNeisserial meningitidis ST269 or ST11 clonal complexes, respectively.

In some aspects, suitable immunogenic compositions of the disclosure arecapable of protecting against infection or disease caused by >50%,suitably >60%, suitably >70%, suitably >80%, suitably >90% of Neisseriameningitidis B strains.

In some aspects, suitable immunogenic compositions of the disclosure arecapable of protecting against infection or disease caused by >50%,suitably >60%, suitably >70%, suitably >80%, suitably >90% of thefollowing group of Neisseria meningitidis B clonal complexes: ST41/44,ST269, ST213.

The antigen capable of generating an antibody response against aNeisseria meningitidis ST269 strain may be any suitable antigen,suitably an antigen capable of generating an immune response whichprotects or ameliorates the infection or disease caused by infectionwith ST269.

In one aspect an antigen which is effective against ST269 is an antigencapable of protecting against infection or disease causedby >50%, >60%, >70%, >80%, >90% of Neisseria meningitidis ST269, moresuitably substantially all of Neisseria meningitidis ST269 clonal types.

The antigen capable of generating an antibody response against aNeisseria meningitidis ST11 strain may be any suitable antigen, suitablyan antigen capable of generating an immune response which protects orameliorates the infection or disease caused by infection with ST11.

In one aspect an antigen which is effective against ST11 is an antigencapable of protecting against infection or disease causedby >50%, >60%, >70%, >80%, >90% of Neisseria meningitidis ST11, moresuitably substantially all of Neisseria meningitidis ST11 clonal types.

Suitable other antigens may be selected from categories of proteinsincluding adhesins, autotransporter proteins, toxins, integral outermembrane proteins and Fe or Zn acquisition proteins.

In one aspect, the disclosure provides immunogenic compositions thatcomprise at least or exactly two, three, four, five, six, seven, eight,nine or ten different Neisseria antigens. Most suitably these antigensare selected from at least or exactly two, three, four or five groups ofproteins selected from the following:

a Neisserial adhesin selected from the group consisting of FhaB, NspAPilC, Hsf, Hap, MafA, MafB, Omp26, NMB 0315, NMB 0995, NMB 1119 andNadA;a Neisserial autotransporter selected from the group consisting of Hsf,Hap, IgA protease, AspA, and NadA;a Neisserial toxin selected from the group consisting of FrpA, FrpC,FrpA/C, VapD, NM-ADPRT and either or both of LPS immunotype L2 and LPSimmunotype L3;a Neisserial Fe or zinc acquisition protein or other metal acquisitionprotein selected from the group consisting of TbpA, TbpB, LbpA, LbpB,HpuA, HpuB, Lipo28 (GNA2132), Sibp, NMB0964, NMB0293, FbpA, Bcp, BfrA,BfrB and P2086 (XthA); anda Neisserial membrane-associated protein, suitably outer membraneprotein, particularly integral outer membrane protein, selected from thegroup consisting of PilQ, OMP85, FhaC, NspA, TbpA, LbpA, TspA, TspB,TdfH, PorB, MItA, HpuB, HimD, H isD, OstA, HIpA (GNA1946), NMB 1124, NMB1162, NMB 1220, NMB 1313, NMB 1953, HtrA, and PldA (OmplA).

The antigens of the present disclosure are all isolated, meaning thatthey are altered by the hand of man. In one aspect they are purified tosome degree, most suitably more than 40, 50, 60, 70, 80, 90, 95 or 99%pure (before combination with the other components of the immunogeniccompositions of the disclosure).

Where a protein is specifically mentioned herein, it is suitably areference to a native, full-length protein, and to its natural variants(i.e. to a native protein obtainable from a Neisserial, suitablymeningococcal strain) but it may also encompass antigenic fragmentsthereof (particularly in the context of subunit vaccines). These arefragments (often specifically described herein) containing or comprisingat least 15 amino acids, suitably at least 20 amino acids, at least 30amino acids, at least 40 amino acids or at least 50 amino acids, takencontiguously from the amino acid sequence of the protein. Antigenicfragments may also be immunogenic fragments. It is further envisagedthat reference to proteins and protein sequences herein includes apolypeptide comprising an immunogenic fragment of 7, 10, 12, 15, 20, 30,40 or 50 (or more) contiguous amino acids from said protein sequence orfrom the amino acid sequence of said protein (optionally wherein saidimmunogenic fragment is capable of eliciting—if necessary when coupledto a protein carrier—an immune response which can recognise said proteinor said protein sequence). In addition, antigenic fragments denotesfragments that are immunologically reactive with antibodies generatedagainst the Neisserial full-length proteins or with antibodies generatedby infection of a mammalian host with Neisseria. Antigenic fragmentsalso includes fragments that when administered at an effective dose,elicit a protective immune response against Neisserial infection, moresuitably it is protective against N. meningitidis and/or N. gonorrhoeaeinfection, most suitably it is protective against N. meningitidisserogroup B infection.

Also included are recombinant fusion proteins of Neisserial proteins, orfragments thereof. These may combine different Neisserial proteins orfragments thereof in the same polypeptide. Alternatively, the disclosurealso includes individual fusion proteins of Neisserial proteins orfragments thereof, as a fusion protein with heterologous sequences suchas a provider of T-cell epitopes or purification tags, for example:p-galactosidase, glutathione-S-transferase, green fluorescent proteins(GFP), epitope tags such as FLAG, myc tag, poly histidine, or viralsurface proteins such as influenza virus haemagglutinin, tetanus toxoid,diphtheria toxoid, CRM197.

Antigens of the disclosure associated with NMB references (or GNAreferences) refer to known reference numbers corresponding to knownsequences (well-known to a skilled person) which can (for example) beaccessed from www.neisseria.org.

Details of the 5 classes of proteins mentioned above are included inWO/2004/014418, hereby incorporated fully by reference.

1. Adhesins—

Adhesins include FhaB (WO98/02547), NadA (J. Exp. Med (2002) 195: 1445;NMB 1994), Hsf also known as NhhA (NMB 0992) (WO99/31132), Hap (NMB1985) (WO99/55873), NspA (WO96/29412), MafA (NMB 0652) and MafB (NMB0643) (Annu Rev Cell Dev Biol. 16; 423-457 (2000); Nature Biotech 20;914-921 (2002)), Omp26 (NMB 0181), NMB 0315, NMB 0995, NMB 1119 and PilC(Mol. Microbiol. 1997, 23; 879-892). These are proteins that areinvolved in the binding of Neisseria to the surface of host cells. Hsfis an example of an adhesin, as well as being an autotranporter protein.Immunogenic compositions of the disclosure may therefore includecombinations of Hsf and other autotransporter proteins where Hsfcontributes in its capacity as an adhesin. These adhesins may be derivedfrom Neisseria meningitidis or Neisseria gonorrhoeae or other Neisserialstrains. The disclosure also includes other adhesins from Neisseria.

FhaB This antigen has been described in WO98/02547 SEQ ID NO 38(nucleotides 3083-9025)—see also NMB0497. The present inventors havefound FhaB to be particularly effectively at inducing anti-adhesiveantibodies alone and in particular with other antigens of thedisclosure. Although full length FhaB could be used, the inventors havefound that particular C-terminal truncates are surprisingly at least aseffective and suitably even more effective in terms of cross-straineffect. Such truncates have also been advantageously shown to be fareasier to clone. FhaB truncates of the disclosure typically correspondto the N-terminal two-thirds of the FhaB molecule, suitably the newC-terminus being situated at position 1200-1600, more suitably atposition 1300-1500, and most suitably at position 1430-1440. Specificembodiments have the C-terminus at 1433 or 1436. Accordingly such FhaBtruncates of the disclosure and vaccines comprising such truncates areindependent aspects of the present disclosure as well as beingcomponents of the combination immunogenic compositions of thedisclosure. The N-terminus may also be truncated by up to 10, 20, 30, 40or 50 amino acids.

2. Autotransporter Proteins

Autotransporter proteins typically are made up of a signal sequence, apassenger domain and an anchoring domain for attachment to the outermembrane. Examples of autotransporter proteins include Hsf (WO99/31132)(NMB 0992), HMW, Hia (van Ulsen et al Immunol. Med. Microbiol. 2001 32;53-64), Hap (NMB 1985) (WO99/55873; van Ulsen et al Immunol. Med.Microbiol. 2001 32; 53-64), UspA, UspA2, NadA (NMB 1994) (Comanducci etal J. Exp. Med. 2002 195; 1445-1454), AspA (Infection and Immunity 2002,70 (8); 4447-4461; NMB 1029), Aida-1 like protein, SSh-2 and Tsh. NadA(J. Exp. Med (2002) 195: 1445) is another example of an autotransporterproteins, as well as being an adhesin. Immunogenic compositions of thedisclosure may therefore include combinations of NadA and adhesins whereNadA contributes in its capacity as an autotransporter protein. Theseproteins may be derived from Neisseria meningitidis or Neisseriagonorrhoeae or other Neisserial strains. The disclosure also includesother autotransporter proteins from Neisseria.

3. Metal Acquisition Proteins Such as Iron and Zinc AcquisitionProteins:

These proteins include Tdfl (NMB0964) (Done J et al Microbiol 2003 andTurner P C et al Microbiol 2001—add full references); TdfH (NmB1497);TbpA (NMB 0461) (WO92/03467, U.S. Pat. No. 5,912,336, WO93/06861 andEP586266), TbpB (NMB 0460) (WO93/06861 and EP586266), LbpA (NMB 1540)(Med Microbiol (1999) 32: 1117), LbpB (NMB 1541) (WO/99/09176), HpuA(U73112.2) (Mol Microbiol. 1997, 23; 737-749), HpuB (NC_(—)003116. 1)(Mol. Microbiol. 1997, 23; 737-749), P2086 also known as XthA (NMB 0399)(13′″ International Pathogenic Neisseria Conference 2002), FbpA (NMB0634), FbpB, BfrA (NMB 1207), BfrB (NMB 1206), Lipo28 also known asGNA2132 (NMB 2132), Sibp (NMB 1882), HmbR, HemH, Bcp (NMB 0750), Iron(III) ABC transporter-permease protein (Tettelin et al Science 287;1809-1815 2000), Iron (III) ABC transporter-periplasmic (Tettelin et alScience 287; 1809-1815 2000), TonB-dependent receptor (NMB 0964 and NMB0293) (Tettelin et al Science 287; 1809-1815 2000) and transferrinbinding protein related protein (Tettelin et al Science 287; 1809-18152000). These proteins may be derived from Neisseria meningitidis,Neisseria gonorrhoeae or other Neisserial strains. The disclosure alsoincludes other metallic ion acquisition proteins from Neisseria.

TbpA interacts with TbpB to form a protein complex on the outer membraneof Neisseria, which binds transferrin. Structurally, TbpA contains anintracellular N-terminal domain with a TonB box and plug domain,multiple transmembrane beta strands linked by short intracellular andlonger extracellular loops.

Two families of TbpB have been distinguished, having a high molecularweight and a low molecular weight respectively. High and low molecularweight forms of TbpB associate with different families of TbpA which aredistinguishable on the basis of homology. Despite being of similarmolecular weight, they are known as the high molecular weight and lowmolecular weight families because of their association with the high orlow molecular weight form of TbpB (Rokbi et al FEMS Microbiol. Lett.

100; 51, 1993). The terms TbpA (high) and TbpA (low) are used to referto these two forms of TbpA, and similarly for TbpB. humanogeniccompositions of the disclosure may comprise TbpA and TbpB fromserogroups A, B, C, Y and W-135 of N. meningitidis as well as ironacquisition proteins from other bacteria including N. gono7rhoeae.Transferrin binding proteins TbpA and TbpB have also been referred to asTbp1 and Tbp2 respectively (Cornelissen et al Infection and Immunity 65;822, 1997).

TbpA contains several distinct regions. For example, in the case of TbpAfrom N. meningitidis strain H44/76, the amino terminal 186 amino acidsform an internal globular domain, 22 beta strands span the membrane,forming a beta barrel structure.

These are linked by short intracellular loops and larger extracellularloops.

Extracellular loops 2, 3 and 5 have the highest degree of sequencevariability and loop 5 is surface exposed. Loops 5 and 4 are involved inligand binding.

Suitable fragments of TbpA include the extracellular loops of TbpA.Using the sequence of TbpA from N. meningitidis strain H44/76, theseloops correspond to amino acids 200-202 for loop 1, amino acids 226-303for loop 2, amino acids 348-395 for loop 3, amino acids 438-471 for loop4, amino acids 512-576 for loop 5, amino acids 609-625 for loop 6, aminoacids 661-671 for loop 7, amino acids 707-723 for loop 8, amino acids769-790 for loop 9, amino acids 814-844 for loop 10 and amino acids872-903 for loop 11. The corresponding sequences, after sequencealignment, in other Tbp proteins would also constitute suitablefragments. Most suitable fragments would include amino acid sequencesconstituting loop 2, loop 3, loop 4 or loop 5 of Tbp.

Where the immunogenic compositions of the disclosure comprise TbpA, itis preferable to include both TbpA (high) and TbpA (low).

Although TbpA is suitably presented in an outer membrane vesicle (OMV)vaccine, it may also be part of a subunit vaccine. For instance,isolated iron acquisition proteins which could be introduced into animmunogenic composition of the disclosure are well known in the art(WO00/25811). They may be expressed in a bacterial host, extracted usingdetergent (for instance 2% Elugent) and purified by affinitychromatography or using standard column chromatography techniques wellknown to the art (Oakhill et al Biochem J. 2002 364; 613-6).

Where TbpA is presented in an OMV vaccine, its expression can beupregulated by genetic techniques discussed herein, or may suitably beupregulated by growth of the parent strain under iron limitationconditions as described below. This process will also result in theupregulation of variable iron-regulated proteins, particularly FrpBwhich may become immunodominant and it is therefore advantageous todownregulate the expression of (and suitably delete the genes encoding)such proteins (particularly FrpB) as described below, to ensure that theimmunogenic composition of the disclosure elicits an immune responseagainst antigens present in a wide range of Neisserial strains. It issuitable to have both TbpA (high) and TbpA (low) present in theimmunogenic composition and this is suitably achieved by combining OMVsderived from two strains, expressing the alternative forms of TbpA.

4. Toxins:

Toxins include FrpA (NMB 0585; NMB 1405), FrpA/C (see below fordefinition), FrpC (NMB 1415; NMB 1405) (WO92/01460), NM-ADPRT (NMB 1343)(13′ h International Pathogenic Neisseria Conference 2002 Masignani etal p135), VapD (NMB 1753), lipopolysaccharide (LPS; also calledlipooligosaccharide or LOS) immunotype L2 and LPS immunotype L3. FrpAand FrpC contain a region which is conserved between these two proteinsand a suitable fragment of the proteins would be a polypeptidecontaining this conserved fragment, suitably comprising amino acids227-1004 of the sequence of FrpA/C. These antigens may be derived fromNeisseria fyzeningitidis or Neisseria gonorrlaoeae or other Neisserialstrains. The disclosure also includes other toxins from Neisseria.

In an alternative embodiment, toxins may include antigens involved inthe regulation of toxicity, for example OstA which functions in thesynthesis of lipopolysaccharides.

FrpA and FrpC Neisseria 7nenngtds encodes two RTX proteins, referred toas FrpA & FrpC secreted upon iron limitation (Thompson et al., (1993) J.Bacteriol. 175: 811-818; Thompson et al., (1993) Infect. Immun. 61:2906-2911). The RTX (Repeat ToXin) protein family have in common aseries of 9 amino acid repeat near their C-termini with the consensus:Leu Xaa Gly Gly Xaa Gly (Asn/Asp) Asp Xaa (LXGGXGN/DDX). The repeats inE. coli HlyA are thought to be the site of Ca2+ binding. MeningococcalFrpA and FrpC proteins, as characterized in strain FAM20, shareextensive amino-acid similarity in their central and C-terminal regionsbut very limited similarity (if any) at the N-terminus.

Moreover, the region conserved between FrpA and FrpC exhibit somepolymorphism due to repetition (13 times in FrpA and 43 times in FrpC)of a 9 amino acid motif.

Immunogenic compositions of the disclosure may comprise the full lengthFrpA and/or FrpC or suitably, a fragment comprising the sequenceconserved between FrpA and FrpC. The conserved sequence is made up ofrepeat units of 9 amino acids.

Immunogenic compositions of the disclosure would suitably comprise morethat three repeats, more than 10 repeats, more than 13 repeats, morethan 20 repeats or more than 23 repeats.

Such truncates have advantageous properties over the full lengthmolecules and composition or vaccines comprising such antigens form anindependent aspect of disclosure as sell as being incorporated in theimmunogenic compositions of the disclosure.

Sequences conserved between FrpA and FrpC are designated FrpA/C andwherever FrpA or FrpC forms a constituent of immunogenic compositions ofthe disclosure, FrpA/C could be advantageously used. Amino acids277-1004 of the FrpA sequence is the suitable conserved region. Theabove sequence can be extended or truncated by up to 1, 3, 5, 7, 10, 15,20, 25, or 30 amino acids at either or both N or C termini.

LPS:

In one aspect of the disclosure L2 LOS is combined with fHbp.

LPS is suitably presented in an outer membrane vesicle (OMV) (suitablywhere the vesicle is extracted with a low percentage detergent, moresuitably 0-0.5%, 0.02-0.4%, 0.04-0. 3%, 0.06-0.2%, 0.08-0.15% or 0.1%,most suitably deoxycholate [DOC]) but may also be part of a subunitvaccine. LPS may be isolated using well known precedure including thehot water-phenol procedure (Wesphal and Jann Meth. Carbo. Chem. 5; 83-911965). See also Galanos et al. 1969, Eur J Biochem 9: 245-249, and Wu etal. 1987, Anal Bio Chem 160: 281-289. LPS may be used plain orconjugated to a source of T-cell epitopes such as tetanus toxoid,Diphtheria toxoid, CRM-197 or OMV outer membrane proteins. Techniquesfor conjugating isolated LOS are also known (see for instance EP 941738incorporated by reference herein).

Where LOS (in particular the LOS of the disclosure) is present in a blebformulation the LOS is suitably conjugated in situ by methods allowingthe conjugation of LOS to one or more outer membrane proteins alsopresent on the bleb preparation (e.g. PorA or PorB in meningococcus).

This process can advantageously enhance the stability and/orimmunogenicity (providing T-cell help) and/or antigenicity of the LOSantigen within the bleb formulation-thus giving T-cell help for theT-independent oligosaccharide immunogen in its most protectiveconformation-as LOS in its natural environment on the surface ofmeningococcal outer membrane. In addition, conjugation of the LOS withinthe bleb can result in a detoxification of the LOS (the Lipid A portionbeing stably buried in the outer membrane thus being less available tocause toxcity). Thus the detoxification methods mentioned herein ofisolating blebs from htrB′ or msbB′ mutants, or by adding non toxicpeptide functional equivalent of polymyxin B [a molecule with highaffinity to Lipid A] to the composition (see WO 93/14115, WO 95/03327,Velucchi et al (1997) J Endotoxin Res 4: 1-12, and EP 976402 for furtherdetails of non-toxic peptide functional equivalents of polymyxinB-particularly the use of the peptide SAEP 2 (of sequence KTKCKFLKKCwhere the 2 cysteines form a disulphide bridge)) may not be required(but which may be added in combination for additional security). Thusthe inventors have found that a composition comprising blebs wherein LOSpresent in the blebs has been conjugated in an intra-bleb fashion toouter membrane proteins also present in the bleb can form the basis of acomposition or vaccine for the treatment or prevention of diseasescaused by the organism from which the blebs have been derived, whereinsuch vaccine is substantially non-toxic and is capable of inducing aT-dependent bactericidal response against LOS in its native environment.

This disclosure therefore further provides such an intra-bleb LOSconjugated meningococcal bleb preparation.

Such bleb preparations may be isolated from the bacterial in question(see WO 01/09350), and then subjected to known conjugation chemistriesto link groups (e.g. NH2 or COOH) on the oligosaccharide portion of LOSto groups (e.g. NH2 or COOH) on bleb outer membrane proteins.Cross-linking techniques using glutaraldehyde, formaldehyde, orglutaraldehyde/formaldehyde mixes may be used, but it is suitable thatmore selective chemistries are used such as EDAC or EDAC/NHS (J. V.Staros, R. W. Wright and D. M. Swingle. Enhancement byN-hydroxysuccinimide of water-soluble carbodiimide-mediated couplingreactions. Analytical chemistry 156: 220-222 (1986); and BioconjugatesTechniques. Greg T. Hermanson (1996) pp 173-176). Other conjugationchemistries or treatments capable of creating covalent links between LOSand protein molecules that could be used are described in EP 941738.

In one aspect the bleb preparations are conjugated in the absence ofcapsular polysaccharide. The blebs may be isolated from a strain whichdoes not produce capsular polysaccharide (naturally or via mutation asdescribed below), or may be purified from most and suitably allcontaminating capsular polysaccharide. In this way, the intra-bleb LOSconjugation reaction is much more efficient.

In one aspect more than 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% ofthe LOS present in the blebs is cross-linked/conjugated.

Intrableb conjugation should suitably incorporate 1, 2 or all 3 of thefollowing process steps: conjugation pH should be greater than pH 7.0,suitably greater than or equal to pH 7.5 (most suitably under pH 9);conditions of 1-5% suitably 2-4% most suitably around 3% sucrose shouldbe maintained during the reaction; NaCl should be minimised in theconjugation reaction, suitably under 0.1M, 0.05M, 0.01M, 0.005M, 0.001M,and most suitably not present at all. All these process features makesure that the blebs remain stable and in solution throughout theconjugation process.

The EDAC/NHS conjugation process is a suitable process for intra-blebconjugation.

EDAC/NHS is suitable to formaldehyde which can cross-link to too high anextent thus adversely affecting filterability. EDAC reacts withcarboxylic acids (such as KDO in LOS) to create an active-esterintermediate. In the presence of an amine nucleophile (such as lysinesin outer membrane proteins such as PorB), an amide bond is formed withrelease of an isourea by-product. However, the efficiency of anEDAC-mediated reaction may be increased through the formation of aSulfo-NHS ester intermediate. The Sulfo-NEIS ester survives in aqueoussolution longer than the active ester formed from the reaction of EDACalone with a carboxylate. Thus, higher yields of amide bond formationmay be realized using this two-stage process.

EDAC/NHS conjugation is discussed in J. V. Staros, R. W. Wright and D.M. Swingle, Enhancement by N-hydroxysuccinimide of water-solublecarbodiimide-mediated coupling reactions. Analytical chemistry 156:220-222 (1986); and Bioconjugates Techniques. Greg T. Hermanson (1996)pp 173-176. In one aspect 0.01-5 mg EDAC/mg bleb is used in thereaction, more suitably 0.05-1 mg EDAC/mg bleb. The amount of EDAC useddepends on the amount of LOS present in the sample which in turn dependson the deoxycholate (DOC) % used to extract the blebs. At low % DOC(e.g. 0.1%), high amounts of EDAC are used (1 mg/mg and beyond), howeverat higher % DOC (e.g. 0.5%), lower amounts of EDAC are used (0.025-0.1mg/mg) to avoid too much inter-bleb crosslinking.

A suitable process of the disclosure is therefore a process forproducing intra-bleb conjugated LOS (suitably meningococcal) comprisingthe steps of conjugating blebs in the presence of EDAC/NHS at a pHbetween pH 7.0 and pH 9.0 (suitably around pH 7.5), in 1-5% (suitablyaround 3%) sucrose, and optionally in conditions substantially devoid ofNaCl (as described above), and isolating the conjugated blebs from thereaction mix.

The reaction may be followed on Western separation gels of the reactionmixture using anti-LOS (e.g. anti-L2 or anti-L3) mAbs to show theincrease of LOS molecular weight for a greater proportion of the LOS inthe blebs as reaction time goes on.

Yields of 99% blebs can be recovered using such techniques.

EDAC was found to be an excellent intra-bleb cross-linking agent in thatit cross-linked LOS to OMP sufficiently for improved LOS T-dependentimmunogenicity, but did not cross link it to such a high degree thatproblems such as poor filterability, aggregation and inter-blebcross-linking occurred. The morphology of the blebs generated is similarto that of unconjugated blebs (by electron microscope). In addition, theabove protocol avoided an overly high cross-linking to take place (whichcan decrease the immunogenicity of protective OMPs naturally present onthe surface of the bleb e.g. TbpA or Hsf).

It is suitable that the meningococcal strain from which the blebs arederived is a mutant strain that cannot produce capsular polysaccharide(e.g. one of the mutant strains described below, in particular siaD′).It is also suitable that immunogenic compositions effective againstmeningococcal disease comprise both an L2 and L3 bleb, wherein the L2and L3 LOS are both conjugated to bleb outer membrane proteins.Furthermore, it is suitable that the LOS structure within the intra-blebconjugated bleb is consistent with it having been derived from anIgtB-meningococcal strain (as described below). Most suitablyimmunogenic compositions comprise intrableb-conjugated blebs: derivedfrom a mutant meningococcal strain that cannot produce capsularpolysaccharide and is IgtB-; comprising L2 and L3 blebs derived frommutant meningococcal strains that cannot produce capsularpolysaccharide; comprising L2 and L3 blebs derived from mutantmeningococcal strains that are IgtB-; or most suitably comprising L2 andL3 blebs derived from mutant meningococcal strains that cannot producecapsular polysaccharide and are IgtB-.

Typical L3 meningococcal strain that can be used for the presentdisclosure is H44/76 menB strain. A typical L2 strain is the B16B6 menBstrain or the 39E meningococcus type C strain.

As stated above, the blebs of the disclosure have been detoxified to adegree by the act of conjugation, and need not be detoxified anyfurther, however further detoxification methods may be used foradditional security, for instance using blebs derived from ameningococcal strain that is htrB′ or msbB- or adding a non-toxicpeptide functional equivalent of polymyxin B [a molecule with highaffinity to Lipid A] (suitably SEAP 2) to the bleb composition (asdescribed above).

In the above way meningococcal blebs and immunogenic compositionscomprising blebs are provided which have as an important antigen LOSwhich is substantially non-toxic, devoid of autoimmunity problems, has aT-dependent character, is present in its natural environment, and iscapable of inducing a bactericidal antibody response against more than90% of meningococcal strains (in the case of L2+L3 compositions).

In one aspect intrableb LOS conjugation should incorporate 1, 2 or all 3of the following process steps: conjugation pH should be greater than pH7.0, suitably greater than or equal to pH 7.5 (most suitably under pH9); conditions of 1-5% suitably 2-4% most suitably around 3% sucroseshould be maintained during the reaction; NaCl should be minimised inthe conjugation reaction, suitably under 0.1M, 0.05M, 0.01M, 0.005M,0.001M, and most suitably not present at all. All these process featuresmake sure that the blebs remain stable and in solution throughout theconjugation process.

Although LOS can be conjugated within blebs to outer membrane proteinsby various techniques and chemistries, the EDAC/NHS conjugation processis a suitable process for intra-bleb conjugation. EDAC/NHS is moresuitable than formaldehyde which can cross-link to too high an extentthus adversely affecting filterability. EDAC reacts with carboxylicacids to create an active-ester intermediate.

In the presence of an amine nucleophile, an amide bond is formed withrelease of an isourea by-product. However, the efficiency of anEDAC-mediated reaction may be increased through the formation of aSulfo-NHS ester intermediate. The Sulfo-NHS ester survives in aqueoussolution longer than the active ester formed from the reaction of EDACalone with a carboxylate. Thus, higher yields of amide bond formationmay be realized using this two-stage process. EDAC/NHS conjugation isdiscussed in J. V. Staros, R. W. Wright and D. M. Swingle. Enhancementby N-hydroxysuccinimide of water-soluble carbodiimide-mediated couplingreactions. Analytical chemistry 156: 220-222 (1986); and BioconjugatesTechniques. Greg T. Hermanson (1996) pp 173-176.

5. Integral Outer Membrane Proteins

Other categories of Neisserial proteins may also be candidates forinclusion in the Neisserial composition or vaccines of the disclosureand may be able to combine with other antigens in a surprisinglyeffective manner. Membrane associated proteins, particularly integralmembrane proteins and most advantageously outer membrane proteins,especially integral outer membrane proteins may be used in thecompositions of the present disclosure. An example of such a protein isPIDA also known as Omp IA (NMB 0464) (WO00/15801) which is a Neisserialphospholipase outer membrane protein. Further examples are TspA (NMB0341) (Infect. Immun. 1999, 67; 3533-3541) and TspB (T-cell stimulatingprotein) (WO 00/03003; NMB 1548, NMB 1628 or NMB 1747).

Further examples include PilQ (NMB 1812) (WO99/61620), OMP85—also knownas D15-(NMB 0182) (WO00/23593), NspA (U52066) (WO96/29412), FhaC(NMB0496 or NMB 1780), PorB (NMB 2039) (Mol. Biol. Evol. 12; 363-370, 1995),HpuB (NC_(—)003116.1), TdfH (NMB 1497) (Microbiology 2001, 147;1277-1290), OstA (NMB 0280), MItA also known as GNA33 and Lipo30(NMB0033), HtrA (NMB 0532; WO 99/55872), HimD (NMB 1302), H isD (NMB1581), HIpA (NMB 1946), NMB 1124, NMB 1162, NMB 1220, NMB 1313, NMB1953, HtrA, TbpA (NMB 0461) (WO92/03467), TdfH (NmB1497) and Tdfl(NMB0964) (see also above under iron and zinc acquisition proteins) andLbpA (NMB 1541).

OMP85 Immunogenic compositions of the disclosure may comprise the fulllength OMP85, suitably as part of an OMV preparation. Fragments of OMP85may also be used in immunogenic compositions of the disclosure, inparticularly, the surface exposed domain of OMP85 made up of amino acidresidues 1-475 or 50-475 is suitably incorporated into a subunitcomponent of the immunogenic compositions of the disclosure. The abovesequence for the surface exposed domain of OMP85 can be extended ortruncated by up to 1, 3, 5, 7, 10, 15, 20, 25, or 30 amino acids ateither or both N or C termini. The signal sequence may be omitted fromthe OMP85 fragment.

OstA OstA functions in the synthesis of lipopolysaccharides and may beconsidered to be a regulator of toxicity. OstA may alternatively beincluded in the toxin category where the toxin category is broadened tocontain regulators of toxicity as well as toxins.

In another aspect the disclosure relates to a polynucleotide encoding aprotein or combination of proteins present in the composition asclaimed. For example, the fHbp may be provided in combination with asecond antigen, either as a polynucleotide encoding a fusion protein oras a polynucleotide encoding polypeptides on the same nucleic acidconstruct (e.g. vector construct)

“Polynucleotide” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions.

In addition, “polynucleotide” refers to triple-stranded regionscomprising RNA or DNA or both RNA and DNA. The term polynucleotide alsoincludes DNAs or RNAs containing one or more modified bases and DNAs orRNAs with backbones modified for stability or for other reasons.“Modified” bases include, for example, tritylated bases and unusualbases such as inosine. A variety of modifications has been made to DNAand RNA; thus, “polynucleotide” embraces chemically, enzymatically ormetabolically modified forms of polynucleotides as typically found innature, as well as the chemical forms of DNA and RNA characteristic ofviruses and cells.

“Polynucleotide” also embraces relatively short polynucleotides, oftenreferred to as oligonucleotides.

Another aspect of the disclosure relates to an immunological/vaccineformulation which comprises one or more polynucleotide(s). Suchtechniques are known in the art, see for example Wolff et al., Science,(1990) 247: 1465-8.

Such composition or vaccines comprise one or more polynucleotide (s)encoding a plurality of proteins corresponding to protein combinationsof the disclosure described above.

The expression of proteins from such polynucleotides may be under thecontrol of a eukaryotic promoter capable of driving expression within amammalian cell. The polynucleotide may additionally comprise sequenceencoding other antigens.

Examples of eukaryotic promoters that could drive the expression includeviral promoters from viruses including adenoviral promoters, retroviralpromoters.

Alternatively, mammalian promoters could be used to drive expression. Ina further aspect the disclosure relates to a method for manufacture ofan improved fHbp based composition or vaccine for prevention oramelioration of Neisseria meningitidis infection or disease, the methodcomprising combining fHbp with a second antigen capable of generating anantibody response against a Neisseria meningitidis L2 immunotype

The composition of the disclosure may be a subunit composition, acomposition comprising antigens in the context of an outer membranevesicle (bleb), or comprise a combination of subunit and outer membranevesicle.

In one aspect the disclosure relates to culturing a Neisseria speciesbacterium producing a NMB0964 polypeptide, wherein the NMB0964polypeptide is produced at a level sufficient to provide for productionof outer membrane vesicles that, when administered to a subject, elicitanti-NMB0964 antibodies; preparing outer membrane vesicles from thecultured bacterium; and combining the outer membrane vesicles with apharmaceutically acceptable excipient to produce an immunogeniccomposition suitable for administration to a subject in combination withan fHbp polypeptide as defined herein.

Certain aspects are described below.

The immunogenic composition or vaccine of the disclosure may be asubunit composition.

Subunit compositions are compositions in which the component orcomponents have been isolated and purified to at least 50%, suitably atleast 60%, 70%, 80%, 90% purity.

Subunit compositions may be in aqueous solution. They may comprisedetergent, suitably non-ionic, zwitterionic or ionic detergent in orderto solubilise hydrophobic portions of the antigens. They may compriselipids so that liposome structures could be formed, allowingpresentation of antigens with a structure that spans a lipid membrane.

N. meningitidis serogroup B (menB) excretes outer membrane blebs insufficient quantities to allow their manufacture on an industrial scale.An outer membrane vesicle may also be prepared via the process ofdetergent extraction of the bacterial cells (see for example EP 11243).

The immunogenic composition of the disclosure may also comprise an outermembrane vesicle preparation suitably having an antigen which has beenbeen upregulated, either recombinantly or by other means includinggrowth under iron-depleted conditions and/or under zinc depletedconditions. Examples of antigens which would be upregulated in such aouter membrane vesicle preparation include; Tdfl, NspA, Hsf, Hap, OMP85,TbpA (high), TbpA (low), LbpA, TbpB, LbpB, PilQ, AspA, Tdfl, TdfH, PorB,HpuB, P2086, NM-ADPRT, MafA, MafB and PldA Such preparations mayoptionally also comprise either or both of LPS immunotype L2 and LPSimmunotype L3.

In one aspect the OMV might comprise antigen which have beendown-regulated.

The manufacture of bleb preparations from Neisserial strains may beachieved by any of the methods well known to a skilled person. In oneaspect the methods disclosed in EP 301992, U.S. Pat. No. 5,597,572, EP11243 or U.S. Pat. No. 4,271,147, Frederikson et al. (NIPH Annals[1991], 14: 67-80), Zollinger et al. (J. Clin. Invest. [1979], 63:836-848), Saunders et al. (Infect. Immun. [1999], 67: 113-119), Drabicket al. (Vaccine [2000], 18: 160-172) or WO 01/09350 (Example 8) areused. In general, OMVs are extracted with a detergent, suitablydeoxycholate, and nucleic acids are optionally removed enzymatically.Purification is achieved by ultracentrifugation optionally followed bysize exclusion chromatography. If 2 or more different blebs of thedisclosure are included, they may be combined in a single container toform a multivalent preparation of the disclosure (although a preparationis also considered multivalent if the different blebs of the disclosureare separate compositions in separate containers which are administeredat the same time [the same visit to a practitioner] to a host).

OMV preparations are usually sterilised by filtration through a 0.2 Fmfilter, and are suitably stored in a sucrose solution (e.g. 3%) which isknown to stabilise the bleb preparations.

Upregulation of proteins within outer membrane vesicle preparations maybe achieved by insertion of an extra copy of a gene into the Neisserialstrain from which the OMV preparation is derived. Alternatively, thepromoter of a gene can be exchanged for a stronger promoter in theNeisserial strain from which the OMV preparation is derived. Suchtechniques are described in WO01/09350. Upregulation of a protein willlead to a higher level of protein being present in OMV compared to thelevel of protein present in OMV derived from unmodified N. meningitidis(for instance strain H44/76). In one aspect the level will be 1.5, 2, 3,4, 5, 7, 10 or 20 times higher.

Where LPS is intended to be an additional antigen in the OMV, a protocolusing a low concentration of extracting detergent (for exampledeoxycholate or DOC) may suitably be used in the OMV preparation methodso as to preserve high levels of bound LPS whilst removing particularlytoxic, poorly bound LPS. The concentration of DOC used is suitably0-0.5% DOC, 0.02-0.4% DOC, 0.04-0.3% DOC more suitably 0.06%-0.2% DOC or0.08-0. 15% DOC most suitably around or exactly 0.1% DOC.

In one aspect OMVs may include native OMVs obtained without detergentextraction, suitably over-expressing an antigen such as fHbp.

“Stronger promoter sequence” refers to a regulatory control element thatincreases transcription for a gene encoding antigen of interest.

“Upregulating expression” refers to any means to enhance the expressionof an antigen of interest, relative to that of the non-modified (i.e.,naturally occurring) bleb.

It is understood that the amount of upregulation will vary depending onthe particular antigen of interest but will not exceed an amount thatwill disrupt the membrane integrity of the bleb. Upregulation of anantigen refers to expression that is at least 10% higher than that ofthe non-modified bleb. In one aspect it is at least 50% higher. Moresuitably it is at least 100% (2 fold) higher. Most suitably it is 3, 4,5, 7, 10, 20 fold higher. Alternatively or additionally, upregulatingexpression may refer to rendering expression non-conditional onmetabolic or nutritional changes, particularly in the case of TbpA,TbpB, LbpA and LbpB. In one aspect the level of expression is assessedwhen blebs have been derived from bacteria grown in iron limitedconditions (for instance in the presence of an iron chelator).

Again for the purpose of clarity, the term “engineering a bacterialstrain to produce less of said antigen” or “down regulation” refers toany means to reduce the expression of an antigen (or the expression of afunctional gene product) of interest, relative to that of thenon-modified (i.e. naturally occurring bleb), suitably by deletion, suchthat expression is at least 10% lower than that of the non-modifiedbleb.

In one aspect it is at least 50% lower and most suitably completelyabsent. If the down regulated protein is an enzyme or a functionalprotein, the downregulation may be achieved by introducing one or moremutations resulting in a 10%, 20%, 50%, 80% or suitably a 100% reductionin enzymatic or functional activity.

The engineering steps required to modulate the expression of Neisserialproteins can be carried out in a variety of ways known to the skilledperson. For instance, sequences (e.g. promoters or open reading frames)can be inserted, and promoters/genes can be disrupted by the techniqueof transposon insertion. For instance, for upregulating a gene'sexpression, a strong promoter could be inserted via a transposon up to 2kb upstream of the gene's initiation codon (more suitably 200-600 bpupstream, most suitably approximately 400 bp upstream). Point mutationor deletion may also be used (particularly for down-regulatingexpression of a gene).

Such methods, however, may be quite unstable or uncertain, and thereforethe engineering step may be suitably performed via a homologousrecombination event. In one aspect, the event takes place between asequence (a recombinogenic region) of at least 30 nucleotides on thebacterial chromosome, and a sequence (a second recombinogenic region) ofat least 30 nucleotides on a vector transformed within the strain. Inone aspect the regions are 40-1000 nucleotides, more suitably 100-800nucleotides, most suitably 500 nucleotides). These recombinogenicregions should be sufficiently similar that they are capable ofhybridising to one another under highly stringent conditions.

Methods used to carry out the genetic modification events hereindescribed (such as the upregulation or downregulation of genes byrecombination events and the introduction of further gene sequences intoa Neisserial genome) are described in WO01/09350. Typical strongpromoters that may be integrated in Neisseria are porA, porB, IgtF, Opa,p110, Ist, and hpuAB. PorA and PorB are suitable as constitutive, strongpromoters. It has been established that the PorB promoter activity iscontained in a fragment corresponding to nucleotides-1 to -250 upstreamof the initiation codon of porB.

Upregulation of Expression of Antigens by Growth in Iron LimitationMedia.

The upregulation of some antigens in an outer membrane vesiclepreparation of the disclosure is suitably achieved by isolating outermembrane vesicles from a parental strain of Neisseria grown under ironlimitation conditions. A low concentration of iron in the medium willresult in increased expression of proteins involved in iron acquisitionincluding TbpA, TbpB, LbpA, LbpB, HpuA, HpuB and P2086. The expressionof these proteins is thereby upregulated without the need forrecombinantly modifying the gene involved, for instance by inserting astronger promoter or inserting an additional copy of the gene. Thedisclosure would also encompass upregulation of iron acquisitionproteins by growth in iron limitation medium where the gene has alsobeen recombinantly modified.

Iron limitation is achieved by the addition of an iron chelator to theculture medium.

Suitable iron chelators include 2,2-Dipyridil, EDDHA(ethylenediamine-di(o-hydroxyphenylacetic acid) and Desferal(deferoxamine mesylate, Sigma). Desferal is the suitable iron chelatorand is added to the culture medium at a concentration of between 10 and100 pM, suitably 25-75 uM, more suitably 50-70 uM, most suitably at6011M. The iron content of medium comes primarily from the yeast extractand soy peptone constituents and the amount present may vary betweenbatches. Therefore different concentrations of Desferal may be optimalto achieve upregulation of iron acquisition proteins in differentbatches of medium. The skilled artisan should easily be able todetermine the optimal concentration. In basic terms, enough ironchelator should be added to the medium to upregulate the expression ofthe desired iron-regulated protein, but not so much so as to adverselyaffect the growth of the bacteria.

In one aspect, upregulation of iron acquisition proteins by growth underiron limited conditions is combined with recombinant upregulation ofother antigens so that the outer membrane vesicle of the disclosure isachieved.

In one aspect zinc limitation may be used to increase expression ofTdfl. This may be achieved for example, using a medium low in Zn²⁺concentration—i.e. under 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4,0.3, 0.2, 0.1, 0.05 or 0.01 μM free Zn²⁺—(such as Roswell Park MemorialInstitute medium 1640 (RPMI) which has around 1.69 μM Zn²⁺ by ICP-MS),or by removing Zn²⁺ in the medium, for instance using a known zincchelator such as TPEN(N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine)—enough should beadded to the medium such that the expression of the NMB0964 ismaximised. Synthetic media such a or Catlin adapted media may also beused (Nutritional profiles of Neisseria gonorrhoeae, Neisseriameningitidis, and Neisseria lactamica in chemically defined media andthe use of growth requirements for gonococcal typing. Catlin B W JInfect Dis. 1973 August; 128(2):178-94.).

An OMV vaccine prepared either in specific culture conditions low inZn2+, or from a mutant N. meningitidis strain engineered to eitherover-express NMB0964 or to remove the Zinc repression mechanism mediatedthrough Zn2+, is enriched in NMB0964, and such OMVs may elicit goodbactericidal antibody responses compared to OMVs which have not beenprepared with these methods.

Down Regulation/Removal of Variable and Non-Protective ImmunodominantAntigen:

Many surface antigens are variable among bacterial strains and as aconsequence are protective only against a limited set of closely relatedstrains. An aspect of this disclosure covers outer membrane vesicles ofthe disclosure in which the expression of other proteins is reduced, or,suitably, gene (s) encoding variable surface protein (s) are deleted.Such deletion results in a bacterial strain producing blebs which, whenadministered in a composition or vaccine, have a stronger potential forcross-reactivity against various strains due to a higher influenceexerted by conserved proteins (retained on the outer membranes) on thevaccinee's immune system. Examples of such variable antigens inNeisseria that may be downregulated in the bleb immunogenic compositionsof the disclosure include PorA, PorB, Opa.

Other types of gene that could be down-regulated or switched off aregenes which, in vivo, can easily be switched on (expressed) or off bythe bacterium. As outer membrane proteins encoded by such genes are notalways present on the bacteria, the presence of such proteins in thebleb preparations can also be detrimental to the effectiveness of thecomposition or vaccine for the reasons stated above. A suitable exampleto down-regulate or delete is Neisseria Opc protein. Anti-Opc immunityinduced by an Opc containing bleb vaccine would only have limitedprotective capacity as the infecting organism could easily become Opc.

For example, these variable or non-protective genes may bedown-regulated in expression, or terminally switched off. This has theadvantage of concentrating the immune system on better antigens that arepresent in low amounts on the outer surface of blebs. By down-regulationit is also meant that surface exposed, variable immunodominant loops ofthe above outer membrane proteins may be altered or deleted in order tomake the resulting outer membrane protein less immunodominant.

Methods for downregulation of expression are disclosed in WO01/09350.

Suitable proteins to be downregulated in the bleb immunogeniccompositions of the disclosure include PorA and OpA; PorA and OpC; OpAand OpC; PorA and OpA and OpC.

Four different Opa genes are known to exist in the meningococcal genome(Aho et al. 1991 Mol. Microbiol. 5: 1429-37), therefore where Opa issaid to be downregulated in expression it is meant that suitably 1, 2, 3or (suitably) all 4 genes present in meningococcus are so downregulated.Such downregulation may be performed genetically as described in WO01/09350 or by seeking readily-found, natural, stable meningococcalstrains that have no or low expression from the Opa loci. Such strainscan be found using the technique described in Poolman et al (1985 J.Med. Micro. 19: 203-209) where cells that are Opa have a differentphenotype to cells expressing Opa which can be seen looking at theappearance of the cells on plates or under a microscope. Once found, thestrain can be shown to be stably Opa by performing a Western blot oncell contents after a fermentation run to establish the lack of Opa.

Where upregulation of some antigens in the outer membrane vesicle isachieved by growth under iron limitation conditions, the variableprotein FrpB (Microbiology 142; 3269-3274, (1996); J. Bacteriol. 181;2895-2901 (1999)) will also be upregulated. The inventors have foundthat it is advantageous to downregulate expression of FrpB under thesecircumstances by downregulating expression of the entire protein asdescribed in WO01/09350 or by deleting variable region (s) of FrpB.

This will ensure that the immune response elicited by the immunogeniccomposition is directed towards antigens that are present in a widerange of strains. Down regulation of FrpB is suitably combined with downregulation of PorA and OpA; PorA and OpC; OpA and OpC; PorA and OpA andOpC in the bleb immunogenic compositions of the disclosure.

In an alternative embodiment of the disclosure, FrpB is downregulated inouter membrane vesicles which have been prepared from Neisseria strainsnot grown under iron limitation conditions.

The blebs in the immunogenic compositions of the disclosure may bedetoxified via methods for detoxification of LPS which are disclosed inWO01/09350. In particular methods for detoxification of LPS of thedisclosure involve the downregulation/deletion of htrB and/or msbBenzymes which are disclosed in WO01/09350. The msbB and htrB genes ofNeisseria are also called IpxL1 and IpxL2, respectively (WO 00/26384)and deletion mutations of these genes are characterised pnenoltypicallyby the msbB-mutant LOS losing one secondary acyl chain), and thehtrB-mutatn LOS losing both secondary acyl chains. WO93/14155 and WO95/03327 describe nontoxic peptide functional equivalents of polymycin Bthat may be used in compositions of the disclosure.

Such methods are suitably combined with methods of bleb extractioninvolving low levels of DOC, suitably 0-0.3% DOC, more suitably0.05%-0.2% DOC, most suitably around or exactly 0.1% DOC.

Further methods of LPS detoxification include adding to the blebpreparations a non-toxic peptide functional equivalent of polymyxin B(suitably SAEP 2) as described above.

Cross-reactive polysaccharides: the isolation of bacterialouter-membrane blebs from encapsulated Gram-negative bacteria oftenresults in the co-purification of capsular polysaccharide. In somecases, this “contaminant” material may prove useful since polysaccharidemay enhance the immune response conferred by other bleb components. Inother cases however, the presence of contaminating polysaccharidematerial in bacterial bleb preparations may prove detrimental to the useof the blebs in a composition or vaccine. For instance, it has beenshown at least in the case of N. meningitidis that the serogroup Bcapsular polysaccharide does not confer protective immunity and issusceptible to induce an adverse auto-immune response in humans.Consequently, outer membrane vesicles of the disclosure may be isolatedfrom a bacterial strain for bleb production, which has been engineeredsuch that it is free of capsular polysaccharide. The blebs will then besuitable for use in humans. A particularly suitable example of such ableb preparation is one from N. meningitidis serogroup B devoid ofcapsular polysaccharide.

This may be achieved by using modified bleb production strains in whichthe genes necessary for capsular biosynthesis and/or export have beenimpaired.

Inactivation of the gene coding for capsular polysaccharide biosynthesisor export can be achieved by mutating (point mutation, deletion orinsertion) either the control region, the coding region or both(suitably using the homologous recombination techniques describedabove), or by any other way of decreasing the enzymatic function of suchgenes. Moreover, inactivation of capsular biosynthesis genes may also beachieved by antisense over-expression or transposon mutagenesis. Asuitable method is the deletion of some or all of the Neisseriameningitidis cps genes required for polysaccharide biosynthesis andexport. For this purpose, the replacement plasmid pMF121 (described inFrosh et al. 1990, Mol. Microbiol. 4: 1215-1218) can be used to delivera mutation deleting the cpsCAD (+galE) gene cluster.

WO 01/09350 discloses plasmid pMF121 (Frosch et al., 1990) has been usedto construct a Neisseria meningitidis B strain lacking the capsularpolysaccharide. This plasmid contains the flanking regions of the genelocus coding for the biosynthesis pathway of the group B polysaccharide(B PS), and the erythromycin resistance gene. Deletion of the B PSresulted in loss of expression of the group B capsular polysaccharide aswell as a deletion in the active copy of galE leading to the synthesisof galactose deficient LPS.

The safety of antibodies raised to L3 or L2 LPS has been questioned, dueto the presence of a structure similar to the lacto-N-neotetraoseoligosaccharide group (Galβl-4GlcNAcβl-3GalRβl-4Glcβl-) present in humanglycosphingolipids. Even if a large number of people has been safelyvaccinated with deoxycholate extracted vesicle vaccines containingresidual amount of L3 LPS (G. Bjune et al, Lancet (1991), 338,1093-1096; GVG. Sierra et al, NIPH ann (1991), 14, 195-210), thedeletion of the terminal part of the LOS saccharidic is advantageous inpreventing any cross-reaction with structures present at the surface ofhuman tissues. In a suitable embodiment, inactivation of the IgtB generesults in an intermediate LPS structure in which the terminal galactoseresidue and the sialic acid are absent (the mutation leaves a4GlcNAcpl-3Galpl-4Glcpl-structure in L2 and L3 LOS). Such intermediatescould be obtained in an L3 and an L2 LPS strain.

An alternative and less suitable (short) version of the LPS can beobtained by turning off the IgtE gene. A further alternative is use ofthe galE mutation (Cloning and molecular analysis of the galE gene ofNeisseria meningitidis and its role in lipopolysaccharide biosynthesis.Jennings M P, van der Ley P, Wilks K E, Maskell D J, Poolman J T, MoxonE R. Mol Microbiol. 1993 October; 10(2):361-9) to obtain both a capsuleminus strain and a LOS with a short alpha-chain (as IgtE mutation).

A further alternative and less suitable version of the LPS can beobtained by turning off the IgtA gene. If such an IgtA-mutation isselected it is suitable to also turn off IgtC expression to prevent thenon-immunogenic L1 immunotype being formed.

LgtB-mutants are most suitable as the inventors have found that this isthe optimal troncation for resolving the safety issue whilst stillretaining an LPS protective oligosaccharide epitope that can stillinduce a bactericidal antibody response.

Therefore, immunogenic compositions of the disclosure further comprisingL2 or L3 preparations (whether purified or in an isolated bleb) ormeningococcal bleb preparations in general are advantageously derivedfrom a Neisserial strain (suitably meningococcal) that has been geneticengineered to permanently downregulate the expression of functional geneproduct from the IgtB, IgtA or IgtE gene, suitably by switching the geneoff, most suitably by deleting all or part of the promoter and/oropen-reading frame of the gene.

Where the above immunogenic compositions of the disclosure are derivedfrom a meningococcus B strain, it is further suitable that the capsularpolysaccharide (which also contains human-like saccharide structures) isalso removed. Although many genes could be switched off to achieve this,the inventors have advantageously shown that it is suitable that thebleb production strain has been genetically engineered to permanentlydownregulate the expression of functional gene product from the siaDgene (i.e. downregulating a-2-8 polysialyltransferase activity),suitably by switching the gene off, most suitably by deleting all orpart of the promoter and/or open-reading frame of the gene. Such aninactivation is described in WO 01/09350. The siaD (also known as synD)mutation is the most advantageous of many mutations that can result inremoving the human-similar epitope from the capsular polysaccharide,because it one of the only mutations that has no effect on thebiosynthesis of the protective epitopes of LOS, thus being advantageousin a process which aims at ultimately using LOS as a protective antigen,and has a minimal effect on the growth of the bacterium. A suitableaspect of the disclosure is therefore a bleb immunogenic preparation asdescribed above which is derived from an IgtE′siaD′, an IgtA-siaD′ or,suitably, an IgtB-siaD-meningococcus B mutant strain. The strain itselfis a further aspect of the disclosure.

Although siaD-mutation is preferable for the above reasons, othermutations which switch off meningococcus B capsular polysaccharidesynthesis may be used.

Thus bleb production strain can be genetically engineered to permanentlydownregulate the expression of functional gene product from one or moreof the following genes: ctrA, ctrB, ctrC, ctrD, synA (equivalent to synXand siaA), synB (equivalent to siaB) or synC (equivalent to siaC) genes,suitably by switching the gene off, most suitably by deleting all orpart of the promoter and/or open-reading frame of the gene. TheIgtE-mutation may be combined with one or more of these mutations. Inone aspect the IgtB-mutation is combined with one or more of thesemutations. A further aspect of the disclosure is therefore a blebimmunogenic preparation as described above which is derived from such acombined mutant strain of meningococcus B. The strain itself is afurther aspect of the disclosure.

The galE mutation may also be used to get both a capsule minus strainand a LOS with a short alpha-chain (as IgtE mutation).

A Neisserial, locus containing various Igt genes, including IgtB andIgtE, and its sequence is known in the art (see M. P. Jennings et al,Microbiology 1999, 145, 3013-3021 and references cited therein, and J.Exp. Med. 180: 2181-2190 [1994]).

Where full-length (non-truncated) LOS is to be used in the finalproduct, it is desirable for LOS not to be sialyated (as such LOSgenerates an immune response against the most dangerous, invasivemeningococcal B strains which are also unsialylated). In such case usinga capsule negative strain which has a deleted synA (equivalent to synXand siaA), synB (equivalent to siaB) or synC (equivalent to siaC) geneis advantageous, as such a mutation also renders menB LOS incapable ofbeing sialylated.

In bleb preparations, particularly in preparations extracted with lowDOC concentrations LPS may be used as an antigen in the immunogeniccomposition of the disclosure. It is however advantageous todownregulate/delete/inactivate enzymatic function of either the IgtE,IgtA (particularly in combination with IgtC), or, suitably, IgtBgenes/gene products in order to remove human like lacto-N-neotetraosestructures. The Neisserial locus (and sequence thereof) comprising theIgt genes for the biosynthesis of LPS oligosaccharide structure is knownin the art (Jennings et al Microbiology 1999 145; 3013-3021 andreferences cited therein, and J. Exp. Med. <BR> <BR> <BR> <BR> <P> 180:2181-2190) [1994]. Downregulation/deletion of IgtB (or functional geneproduct) is suitable since it leaves the LPS protective epitope intact.

In N. meningitidis serogroup B bleb preparations of the disclosure, thedownregulation/deletion of both siaD and IgtB is suitable, (although acombination of IgtB- with any of ctrA-, ctrB-, ctrC-, ctrD′, synA-(equivalent to synX- and siaA-), synB- (equivalent to siam-) or synC-(equivalent to siam-) in a meningococcus B bleb production strain mayalso be used) leading to a bleb preparation with optimal safety and LPSprotective epitope retention.

A further aspect of the disclosure is therefore a bleb immunogenicpreparation as described above which is derived from such a combinedmutant strain of meningococcus B. The strain itself is a further aspectof the disclosure.

Immunogenic composition of the disclosure may comprise at least, one,two, three, four or five different outer membrane vesicle preparations.Where two or more OMV preparations are included, at least one antigen ofthe disclosure is upregulated in each OMV. Such OMV preparations may bederived from Neisserial strains of the same species and serogroup orsuitably from Neisserial strains of different class, serogroup,serotype, subserotype or immunotype. For example, an immunogeniccomposition may comprise one or more outer membrane vesicle preparation(s) which contains LPS of immunotype L2 and one or more outer membranevesicle preparation which contains LPS of immunotype L3. L2 or L3 OMVpreparations are suitably derived from a stable strain which has minimalphase variability in the LPS oligosaccharide synthesis gene locus.

The immunogenic compositions of the disclosure may also comprise both asubunit composition and an outer membrane vesicle. There are severalantigens that are particularly suitable for inclusion in a subunitcomposition due to their solubility.

Examples of such proteins include; Tdfl, FhaB, NspA, passenger domain ofHsf, passenger domain of Hap, passenger domain of AspA, AspA, OMP85,FrpA, FrpC, TbpB, LbpB, PilQ. The outer membrane vesicle preparationwould have at least one different antigen selected from the followinglist which has been recombinantly upregulated in the outer membranevesicle: NspA, Hsf, Hap, OMP85, TbpA (high), TbpA (low), LbpA, TbpB,LbpB, NadA, TspA, TspB, PilC, PilQ, TdfH, PorB, HpuB, P2086, NM-ADPRT,MafA, MafB and PIdA; and optionally comprise either or both of LPSimmunotype L2 and LPS immunotype L3.

Another aspect of the disclosure is a genetically engineered Neisserialstrain from which an outer membrane vesicle of the disclosures(optionally having at least two proteins of the disclosure recombinantlyupregulated, as described above) may be derived. Such Neisserial strainsmay be Neisseria meningitidis or Neisseria gonorrhoeae.

The strain may also have been engineered (as described above) todownregulate the expression of other Neisserial proteins including theexpression of one, two, three, four, five, six, seven or eight of LgtB,LgtE, SiaD, OpC, OpA, PorA, FrpB, msbB and HtrB. Suitable combinationsfor downregulation include down regulation (suitably deletion) of atleast LgtB and SiaD, downregulation of at least PorA and OpC,downregulation of at least PorA and OpA and downregulation of at leastPorA, OpA and OpC.

In accordance with the above disclosure concerning bleb production,further aspects of the disclosure includes methods of making theimmunogenic composition or vaccine of the disclosure. These include amethod comprising a step of mixing together the chimaeric protein of thedisclosure with an isolated antigens or proteins from Neisseria, whichmay be present in the form of blebs derived from the Neisserial strainsof the disclosure, to make an immunogenic composition of the disclosure,and a method of making the composition or vaccine of the disclosurecomprising a step of combining the immunogenic composition of thedisclosure with a pharmaceutically acceptable carrier.

Also included in the disclosure are methods of making the immunogeniccomposition of the disclosure comprising a step of isolating outermembrane vesicles comprising the chimaeric protein from a Neisserialculture. Such a method may involve a further step of combining at leasttwo outer membrane vesicle preparations, in one aspect wherein at leastone outer membrane vesicle preparation contains LPS of immunotype L2 andat least one outer membrane vesicle preparation contains LPS ofimmunotype L3. The disclosure also includes such methods wherein theouter membrane vesicles are isolated by extracting with a concentrationof DOC of 0-0.5%. DOC concentrations of 0.3%-0.5% are used to minimiseLPS content. In OMV preparations where LPS is to be conserved as anantigen, DOC concentrations of 0-0.3%, suitably 0.05%-0.2%, mostsuitably of about 0.1% are used for extraction.

The immunogenic composition of the disclosure may further comprisebacterial capsular polysaccharides or oligosaccharides. The capsularpolysaccharides or oligosaccharides may be derived from one or more of:Neisseria meningitidis serogroup A, C, Y, and/or W-135, Haemophilusinfluenzae b, Streptococcus pneumonia, Group A Streptococci, Group BStreptococci, Staphylococcus aureus and Staphylococcus epidermidis.

A further aspect of the disclosure are composition or vaccinecombinations comprising the antigenic composition of the disclosure withother antigens which are advantageously used against certain diseasestates including those associated with viral or Gram positive bacteria.

In one suitable combination, the antigenic compositions of thedisclosure are formulated with 1, 2, 3 or suitably all 4 of thefollowing meningococcal capsular polysaccharides or oligosaccharideswhich may be plain or conjugated to a protein carrier: A, C, Y or W-135.In one aspect the immunogenic compositions of the disclosure areformulated with A and C; or C; or C and Y. Such a composition or vaccinecontaining proteins from N. meningitidis, suitably serogroup B may beadvantageously used as a global meningococcus vaccine.

In a further suitable embodiment, the antigenic compositions of thedisclosure, suitably formulated with 1, 2, 3 or all 4 of the plain orconjugated meningococcal capsular polysaccharides or oligosaccharides A,C, Y or W-135 (as described above), are formulated with a conjugated H.influenzae b capsular polysaccharide or oligosaccharides, and/or one ormore plain or conjugated pneumococcal capsular polysaccharides oroligosaccharides. Optionally, the vaccine may also comprise one or moreprotein antigens that can protect a host against Streptococcuspneumoniae infection. Such a vaccine may be advantageously used as aglobal meningitis vaccine.

In a still further suitable embodiment, the immunogenic composition ofthe disclosure is formulated with capsular polysaccharides oroligosaccharides derived from one or more of Neisseria meningitidis,Haemophilus influenzae b, Streptococcus pneumoniae, Group AStreptococci, Group B Streptococci, Staphylococcus aureus orStaphylococcus epidermidis. The pneumococcal capsular polysaccharideantigens are suitably selected from serotypes 1, 2, 3, 4, 5, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F(most suitably from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and23F). A further suitable embodiment would contain the PRP capsularpolysaccharides of Haemophilus influenzae. A further suitable embodimentwould contain the Type 5, Type 8 or 336 capsular polysaccharides ofStaphylococcus aureus. A further suitable embodiment would contain theType I, Type II or Type III capsular polysaccharides of Staphylococcusepidermidis. A further suitable embodiment would contain the Type Ia,Type Ic, Type II or Type III capsular polysaccharides of Group Bstreptococcus. A further suitable embodiment would contain the capsularpolysaccharides of Group A streptococcus, suitably further comprising atleast one M protein and more suitably multiple types of M protein.

Such capsular polysaccharides of the disclosure may be unconjugated orconjugated to a carrier protein such as tetatus toxoid, tetanus toxoidfragment C, diphtheria toxoid, CRM197, pneumolysin, Protein D (U.S. Pat.No. 6,342,224). The polysaccharide conjugate may be prepared by anyknown coupling technique. For example the polysaccharide can be coupledvia a thioether linkage. This conjugation method relies on activation ofthe polysaccharide with 1-cyano-4-dimethylamino pyridiniumtetrafluoroborate (CDAP) to form a cyanate ester. The activatedpolysaccharide may thus be coupled directly or via a spacer group to anamino group on the carrier protein. In one aspect, the cyanate ester iscoupled with hexane diamine and the amino-derivatised polysaccharide isconjugated to the carrier protein using heteroligation chemistryinvolving the formation of the thioether linkage. Such conjugates aredescribed in PCT published application WO93/15760 Uniformed ServicesUniversity.

The conjugates can also be prepared by direct reductive aminationmethods as described in U.S. Pat. No. 4,365,170 (Jennings) and U.S. Pat.No. 4,673,574 (Anderson). Other methods are described in EP-0-161-188,EP-208375 and EP-0-477508. A further method involves the coupling of acyanogen bromide activated polysaccharide derivatised with adipic acidhydrazide (ADH) to the protein carrier by Carbodiimide condensation (ChuC. et al Infect. Immunity, 1983 245 256). Where oligosaccharides areincluded, it is suitable that they be conjugated.

Suitable pneumococcal proteins antigens are those pneumococcal proteinswhich are exposed on the outer surface of the pneumococcus (capable ofbeing recognised by a host's immune system during at least part of thelife cycle of the pneumococcus), or are proteins which are secreted orreleased by the pneumococcus.

Most suitably, the protein is a toxin, adhesin, 2-component signaltranducer, or lipoprotein of Streptococcus pneumoniae, or fragmentsthereof. Particularly suitable proteins include, but are not limited to:pneumolysin (suitably detoxified by chemical treatment or mutation)[Mitchell et al. Nucleic Acids Res. 1990 Jul. 11; 18 (13): 4010“Comparison of pneumolysin genes and proteins from Streptococcuspneumoniae types 1 and 2.”, Mitchell et al. Biochim Biophys Acta 1989Jan. 23; 1007 (1): 67-72 “Expression of the pneumolysin gene inEscherichia coli: rapid purification and biological properties.”, WO96/05859 (A. Cyanamid), WO 90/06951 (Paton et al), WO 99/03884 (NAVA)];PspA and transmembrane deletion variants thereof (U.S. Pat. No.5,804,193-Briles et al.); PspC and transmembrane deletion variantsthereof (WO 97/09994-Briles et al); PsaA and transmembrane deletionvariants thereof (Berry & Paton, Infect Immun 1996 December; 64 (12):5255-62 “Sequence heterogeneity of PsaA, a 37-kilodalton putativeadhesin essential for virulence of Streptococcus pneumonia”);pneumococcal choline binding proteins and transmembrane deletionvariants thereof; CbpA and transmembrane deletion variants thereof (WO97/41151; WO 99/51266); Glyceraldehyde-3-phosphate-dehydrogenase(Infect. Immun. 1996 64: 3544); HSP70 (WO 96/40928); PcpA (Sanchez-Beatoet al. FEMS Microbiol Lett 1998, 164: 207-14); M like protein, (EP0837130) and adhesin 18627, (EP 0834568). Further suitable pneumococcalprotein antigens are those disclosed in WO 98/18931, particularly thoseselected in WO 98/18930 and PCT/US99/30390.

The immunogenic composition/vaccine of the disclosure may alsooptionally comprise outer membrane vesicle preparations made from otherGram negative bacteria, for example Moraxella catarrhalis or Haemophilusinfluenzae.

Immunogenic compositions of the disclosure may further comprise OMVpreparations derived from Moraxella catarrhalis. Engineered OMVpreparations can be derived from Moraxella catarrhalis as described inWO01/09350. One or more of the following genes (encoding protectiveantigens) are suitable for upregulation: OMP106 (WO 97/41731 & WO96/34960), HasR (PCT/EP99/03824), PilQ (PCT/EP99/03823), OMP85(PCT/EP00/01468), lipo06 (GB 9917977.2), lipolO (GB 9918208.1), lipoll(GB 9918302.2), lipol8 (GB 9918038.2), P6 (PCT/EP99/03038), ompCD, CopB(Helminen M E, et al (1993) Infect. Immun. 61: 2003-2010), D15(PCT/EP99/03822), OmplAl (PCT/EP99/06781), Hly3 (PCT/EP99/03257), LbpAand LbpB (WO 98/55606), TbpA and TbpB (WO 97/13785 & WO 97/32980), OmpE,UspA1 and UspA2 (WO 93/03761), and Omp21. They are also suitable asgenes which may be heterologously introduced into other Gram-negativebacteria.

One or more of the following genes are suitable for downregulation:CopB, OMP106, OmpBl, TbpA, TbpB, LbpA, and LbpB.

One or more of the following genes are suitable for downregulation:htrB, msbB and IpxK.

One or more of the following genes are suitable for upregulation: pmrA,pmrB, pmrE, and pmrF.

Immunogenic compositions of the disclosure may further comprise OMVpreparations derived from Haemophilus influenzae. Engineered OMVpreparations can be derived from Haemophilus influenzae as described inWO01/09350. One or more of the following genes (encoding protectiveantigens) are suitable for upregulation: D15 (WO 94/12641), P6 (EP281673), TbpA (WO96/40929; WO95/13370), TbpB (WO96/40929; WO95/13370),P2, P5 (WO 94/26304), OMP26 (WO 97/01638), HMW1, HMW2, HMW3, HMW4, Hia,Hsf, Hap, Hin47, and Hif (all genes in this operon should be upregulatedin order to upregulate pilin). They are also suitable as genes which maybe heterologously introduced into other Gram-negative bacteria.

One or more of the following genes are preferred for downregulation: P2,P5, Hif, IgAl-protease, HgpA, HgpB, HMW1, HMW2, Hxu, htrB, msbB andIpxK.

One or more of the following genes are preferred for upregulation: pmrA,pmrB, pmrE, and pmrF.

The immunogenic composition/vaccine of the disclosure may alsooptionally comprise antigens providing protection against one or more ofDiphtheria, tetanus and Bordetella pertussis infections. The pertussiscomponent may be killed whole cell B. pertussis (Pw) or acellularpertussis (Pa) which contains at least one antigen (suitably 2 or all 3)from PT, FHA and 69 kDa pertactin. Typically, the antigens providingprotection against Diphtheria and tetanus would be Diphtheria toxoid andtetanus toxoid. The toxoids may chemically inactivated toxins or toxinsinactivated by the introduction of point mutations.

The immunogenic composition/vaccine may also optionally comprise one ormore antigens that can protect a host against non-typeable Haemophillusinfluenzae, RSV and/or one or more antigens that can protect a hostagainst influenza virus. Such a vaccine may be advantageously used as aglobal otitis media vaccine.

Suitable non-typeable H. influenzae protein antigens include Fimbrinprotein (U.S. Pat. No. 5,766,608) and fusions comprising peptidestherefrom (eg LB1 Fusion) (U.S. Pat. No. 5,843,464-Ohio State ResearchFoundation), OMP26, P6, protein D, TbpA, TbpB, Hia, Hmwl, Hmw2, Hap, andD15.

Suitable influenza virus antigens include whole, live or inactivatedvirus, split influenza virus, grown in eggs or MDCK cells, or Vero cellsor whole flu virosomes (as described by R. Gluck, Vaccine, 1992, 10,915-920) or purified or recombinant proteins thereof, such as HA, NP,NA, or M proteins, or combinations thereof.

Suitable RSV (Respiratory Syncytial Virus) antigens include the Fglycoprotein, the G glycoprotein, the HN protein, the M protein orderivatives thereof.

Immunogenic compositions of the disclosure may include proteins ofMoraxella catarrhalis include TbpA (WO97/13785; WO99/52947), TbpB(WO97/13785; WO99/52947; Mathers et al FEMS Immunol Med Microbiol 199719; 231-236; Myers et al Infect Immun 1998 66; 4183-4192), LbpA, LbpB(Du et al Infect Immun 1998 66; 3656-3665), UspAl, UspA2 (Aebi et alInfect Immun. 1997 65; 4367-4377), OMP106 (U.S. Pat. No. 6,214,981),Ton-B dependent receptor (WO00/78968), CopB (Sethi et al Infect. Immun.1997 65; 3666-3671), and HasR receptor (WO00/78968); proteins ofHaemophilus influenzae include HMW (St Geme et al Infect Immun 1998 66;364-368), Hia (St Geme et al J. Bacteriol. 2000 182; 6005-6013), Tbpl(WO96/40929; WO95/13370), Tbp2 (WO96/40929; WO95/13370; Gray-Owen et alInfect Immun 1995 63; 1201-1210), LbpA, LbpB (Schryvers et al 1989, 29:121-130), HasR, TonB-dependent receptor (Fleishmann et al Science 1995269; 496-512), hemoglobin-binding protein, HhuA (Cope et al Infect Immun2000 68; 4092-4101), HgpA (Maciver et al Infect Immun 1996 64;3703-3712), HgbA, HgbB and HgbC (Jin et al Infect Immun 1996 64;3134-3141), HxuA (Cope et al Mol Microbiol 1994 13; 863-873), HxuC (Copeet al Infect Immun 2001 69; 2353-2363); proteins from Neisseria meni7ngitidis include Tbpl, Tbp2, FbpA, FbpB, BfrA, BfrB (Tettelin et alScience 2000 287; 1809-1815), LbpA, LbpB and HmbR.

In one aspect the disclosure also relates to a pharmaceuticalcomposition comprising antigens and immunogenic compositions of thedisclosure in combination with a pharmaceutically acceptable excipient.Suitable excipients are well known in the art. Suitable excipients aretypically large, slowly metabolised macromolecules such as proteins,saccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, sucrose (Paoletti et al., 2001, Vaccine,19:2118), trehalose (WO 00/56365), lactose and lipid aggregates (such asoil droplets or liposomes). Such carriers are well known to those ofordinary skill in the art. The vaccines may also contain diluents, suchas water, saline, glycerol, etc. Additionally, auxiliary substances,such as wetting or emulsifying agents, pH buffering substances, and thelike, may be present. Sterile pyrogen-free, phosphate bufferedphysiologic saline is a typical carrier. A thorough discussion ofpharmaceutically acceptable excipients is available in referenceGennaro, 2000, Remington: The Science and Practice of Pharmacy,20.sup.th edition, ISBN:0683306472.

In a further aspect the disclosure relates to a pharmaceuticalcomposition comprising an fHbp polypeptide antigen and a second antigencapable of generating an antibody response against a Neisserialmeningitidis L2 immunotype, in combination with a pharmaceuticallyacceptable excipient.

One embodiment of the disclosure is the formulation of the antigens andimmunogenic compositions of the disclosure in a vaccine apharmaceutically acceptable excipient.

Vaccine preparation is generally described in Vaccine Design (“Thesubunit and adjuvant approach” (eds Powell M. F. & Newman M. J.) (1995)Plenum Press New York).

The development of effective vaccines requires reliable tests forestablishing whether an effective immune response has been elicited invaccinated individuals. For N. meningitides, Serum Bactericidal Activity(SBA) assays may be used to determine suitable antigens for inclusion inany vaccine, and suitably a four-fold increase in SBA may be accepted asa surrogate for protection.

Thus, a dose that would induce a 4-fold increase in SBA may be acceptedas a protective dose or an effective amount. In particular, the vaccinesor immunogenic compositions of the invention may comprise a human doseof (or of more than) 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08,0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 μg of each of the recited (isolated and/or purified) antigensin the composition.

A pharmaceutical composition or vaccine of the disclosure may alsocomprise an adjuvant.

Suitable adjuvants include an aluminium salt such as aluminum hydroxidegel (alum) or aluminium phosphate, but may also be a salt of calcium(particularly calcium carbonate), iron or zinc, or may be an insolublesuspension of acylated tyrosine, or acylated sugars, cationically oranionically derivatised polysaccharides, or polyphosphazenes.

Suitable Thl adjuvant systems that may be used include, Monophosphoryllipid A, particularly 3-de-O-acylated monophosphoryl lipid A, and acombination of monophosphoryl lipid A, suitably 3-de-O-acylatedmonophosphoryl lipid A (3D-MPL) together with an aluminium salt(suitably aluminium phosphate). An enhanced system involves thecombination of a monophosphoryl lipid A and a saponin derivativeparticularly the combination of QS21 and 3D-MPL as disclosed in WO94/00153, or a less reactogenic composition where the QS21 is quenchedwith cholesterol as disclosed in WO96/33739. A particularly potentadjuvant formulation involving QS21 3D-MPL and tocopherol in an oil inwater emulsion is described in WO95/17210 and is a suitable formulation.

The vaccine may comprise a saponin, more suitably QS21. It may alsocomprise an oil in water emulsion and tocopherol. Unmethylated CpGcontaining oligo nucleotides (WO 96/02555) are also preferentialinducers of a TH1 response and are suitable for use in the presentdisclosure.

Another aspect of the disclosure is a method of preparing animmunoglobulin for use in prevention or treatment of Neisserialinfection comprising the steps of immunizing a recipient with thevaccine of the disclosure and isolating immunoglobulin from therecipient. An immunoglobulin prepared by this method is a further aspectof the disclosure. A pharmaceutical composition comprising theimmunoglobulin of the disclosure and a pharmaceutically acceptablecarrier is a further aspect of the disclosure which could be used in themanufacture of a medicament for the treatment or prevention ofNeisserial disease. A method for treatment or prevention of Neisserialinfection comprising a step of administering to a patient an effectiveamount of the pharmaceutical preparation of the disclosure is a furtheraspect of the disclosure.

Inocula for polyclonal antibody production are typically prepared bydispersing the antigenic composition in a physiologically tolerablediluent such as saline or other adjuvants suitable for human use to forman aqueous composition. An immunostimulatory amount of inoculum isadministered to a mammal and the inoculated mammal is then maintainedfor a time sufficient for the antigenic composition to induce protectiveantibodies.

The antibodies can be isolated to the extent desired by well knowntechniques such as affinity chromatography (Harlow and Lane Antibodies;a laboratory manual 1988).

Antibodies can include antiserum preparations from a variety of commonlyused animals e.g. goats, primates, donkeys, swine, horses, guinea pigs,rats or man. The animals are bled and serum recovered.

An immune globulin produced in accordance with the present disclosurecan include whole antibodies, antibody fragments or subfragments.Antibodies can be whole immunoglobulins of any class e.g. IgG, IgM, IgA,IgD or IgE, chimeric antibodies or hybrid antibodies with dualspecificity to two or more antigens of the disclosure. They may also befragments e.g. F (ab′)2, Fab′, Fab, Fv and the like including hybridfragments. An immune globulin also includes natural, synthetic orgenetically engineered proteins that act like an antibody by binding tospecific antigens to form a complex.

A vaccine of the present disclosure can be administered to a recipientwho then acts as a source of immune globulin, produced in response tochallenge from the specific vaccine. A subject thus treated would donateplasma from which hyperimmune globulin would be obtained viaconventional plasma fractionation methodology. The hyperimmune globulinwould be administered to another subject in order to impart resistanceagainst or treat Neisserial infection. Hyperimmune globulins of thedisclosure are particularly useful for treatment or prevention ofNeisserial disease in infants, immune compromised individuals or wheretreatment is required and there is no time for the individual to produceantibodies in response to vaccination.

An additional aspect of the disclosure is a pharmaceutical compositioncomprising two of more monoclonal antibodies (or fragments thereof;suitably human or humanized) reactive against at least two constituentsof the immunogenic composition of the disclosure, which could be used totreat or prevent infection by Gram negative bacteria, suitablyNeisseria, more suitably Neisseria meningitidis or Neisseria gonorrhoeaeand most suitably Neisseria meningitidis serogroup B.

Such pharmaceutical compositions comprise monoclonal antibodies that canbe whole immunoglobulins of any class e.g. IgG, IgM, IgA, IgD or IgE,chimeric antibodies or hybrid antibodies with specificity to two or moreantigens of the disclosure. They may also be fragments e.g. F (ab′)2,Fab′, Fab, Fv and the like including hybrid fragments.

Methods of making monoclonal antibodies are well known in the art andcan include the fusion of splenocytes with myeloma cells (Kohler andMilstein 1975 Nature 256; 495; Antibodies-a laboratory manual Harlow andLane 1988). Alternatively, monoclonal Fv fragments can be obtained byscreening a suitable phage display library (Vaughan T J et al 1998Nature Biotechnology 16; 535). Monoclonal antibodies may be humanized orpart humanized by known methods.

Another aspect of the disclosure involves a method for treatment orprevention of Neisserial disease comprising administering a protectivedose (or effective amount) of the vaccine of the disclosure to a host inneed thereof.

The disclosure also includes a use of the immunogenic composition of thedisclosure in the preparation of a medicament for treatment orprevention of Neisserial infection or disease, and to an immunogeniccomposition as described herein for treatment or prevention ofNeisserial meningitidis infection or disease.

In one aspect the prevention is prevention against menB infection and/ordisease.

The host is suitably a human host.

The vaccine preparation of the present disclosure may be used to protector treat a mammal susceptible to infection, by means of administeringsaid vaccine via systemic or mucosal route. These administrations mayinclude injection via the intramuscular, intraperitoneal, intradermal orsubcutaneous routes; or via mucosal administration to theoral/alimentary, respiratory, genitourinary tracts. Thus one aspect ofthe present disclosure is a method of immunizing a human host against adisease caused by infection of a gram-negative bacteria, which methodcomprises administering to the host an immunoprotective dose of thepreparation of the present disclosure.

The amount of antigen in each vaccine dose is selected as an amountwhich induces an immunoprotective response without significant, adverseside effects in typical vaccines. Such amount will vary depending uponwhich specific immunogen is employed and how it is presented. Generally,it is expected that each dose will comprise l-100 pg of protein antigenor OMV preparation, suitably 5-50 pg, and most typically in the range5-25 ut.

An optimal amount for a particular vaccine can be ascertained bystandard studies involving observation of appropriate immune responsesin subjects.

Following an initial vaccination, subjects may receive one or severalbooster immunizations adequately spaced.

The vaccines of the disclosure are suitably immunoprotective andnon-toxic and suitable for paediatric or adolescent use.

By paediatric use it is meant use in infants less than 4 years old.

By immunoprotective it is meant that the SBA and/or animal protectionmodel and/or adhesion blocking assay described above are satisfactorilymet.

By non-toxic it is meant that there is no more than a satisfactory levelof endotoxin activity in the vaccine as measured by the well-known LALand pyrogenicity assays.

The efficacy of vaccines can be assessed through a variety of assays.Protection assays in animal models are well known in the art.Furthermore, serum bactericidal assay (SBA) is the most commonly agreedimmunological marker to estimate the efficacy of a meningococcal vaccine(Perkins et al. J Infect Dis. 1998, 177: 683-691).

Such a synergistic response may be characterised by the SBA elicited bythe combination of antigens being at least 50%, two times, three times,suitably four times, five times, six times, seven times, eight times,nine times and most suitably ten times higher than the SBA elicited byeach antigen separately. In one aspect SBA is measured against ahomologous strain from which the antigens are derived and suitably alsoagainst a panel of heterologous strains. (See below for a representativepanel for instance BZ10 (B: 2b: P1.2) belonging to the A-4 cluster;B16B6 (B: 2a: P1.2) belonging to the ET-37 complex; H44/76 (B: 15: P1.7,16), NZ124/98 (B:4:P1.7-2, 4:L3 ST-44 complex), and 760676 (B:2a:P1.5,2:L2 ST-11 complex). SBA is the most commonly agreed immunologicalmarker to estimate the efficacy of a meningococcal vaccine (Perkins etal. J Infect Dis. 1998, 177: 683-691). Satisfactory SBA can beacertained by any known method. SBA can be carried out using seraobtained from animal models or from human subjects.

A suitable method of conducting SBA with human sera is the following. Ablood sample is taken prior to the first vaccination, two months afterthe second vaccination and one month after the third vaccination (threevaccinations in one year being a typical human primary vaccinationschedule administered at, for instance, 0, 2 and 4 months, or 0, 1 and 6months). Such human primary vaccination schedules can be carried out oninfants under 1 year old (for instance at the same time as Hibvaccinations are carried out) or 2-4 year olds or adolescents may alsobe vaccinated to test SBA with such a primary vaccination schedule. Afurther blood sample may be taken 6 to 12 months after primaryvaccination and one month after a booster dose, if applicable.

SBA will be satisfactory for an antigen or bleb preparation withhomologous bactericidal activity if one month after the third vaccinedose (of the primary vaccination schedule) (in 2-4 year olds oradolescents, but suitably in infants in the first year of life) thepercentage of subjects with a four-fold increase in terms of SBA(antibody dilution) titre (compared with pre-vaccination titre) againstthe strain of meningococcus from which the antigens of the disclosurewere derived is greater than 30%, suitably greater than 40%, moresuitably greater than 50%, and most suitably greater than 60% of thesubjects.

Of course an antigen or bleb preparation with heterologous bactericidalactivity can also constitute bleb preparation with homologousbactericidal activity if it can also elicit satisfactory SBA against themeningococcal strain from which it is derived.

SBA will be satisfactory for an antigen or bleb preparation withheterologous bactericidal activity if one month after the third vaccinedose (of the primary vaccination schedule) (in 2-4 year olds oradolescents, but suitably in infants in the first year of life) thepercentage of subjects with a four-fold increase in terms of SBA(antibody dilution) titre (compared with pre-vaccination titre) againstthree heterologous strains of meningococcus is greater than 20%,suitably greater than 30%, more suitably greater than 35%, and mostsuitably greater than 40% of the subjects. Such a test is a goodindication of whether the antigen or bleb preparation with heterologousbactericidal activity can induce cross-bactericidal antibodies againstvarious meningococcal strains. The three heterologous strains shouldsuitably have different electrophoretic type (ET)-complex or multilocussequence typing (MLST) pattern (see Maiden et al. PNAS USA 1998, 95:3140-5) to each other and suitably to the strain from which the antigenor bleb preparation with heterologous bactericidal activity is made orderived. A skilled person will readily be able to determine threestrains with different ET-complex which reflect the genetic diversityobserved amongst meningococci, particularly amongst meningococcus type Bstrains that are recognised as being the cause of significant diseaseburden and/or that represent recognised MenB hyper-virulent lineages(see Maiden et al. supra). For instance three strains that could be usedare the following: BZ10 (B: 2b: P1.2) belonging to the A-4 cluster;B16B6 (B: 2a: P1.2) belonging to the ET-37 complex; and H44/76 (B: 15:P1.7, 16) belonging to the ET-5 complex, or any other strains belongingto the same ET/Cluster. Such strains may be used for testing an antigenor bleb preparation with heterologous bactericidal activity made orderived from, for instance, meningococcal strain CU385 (B: 4: P1.15)which belongs to the ET-5 complex.

Another sample strain that could be used is from the Lineage 3 epidemicclone (e.g. NZ124 [B: 4: P1.7, 4]). Another ET-37 strain is NGP165 (B:2a: P1.2).

Processes for measuring SBA activity are known in the art. For instancea method that might be used is described in WO 99/09176 in Example 100.In general terms, a culture of the strain to be tested is grown(suitably in conditions of iron depletion-by addition of an ironchelator such as EDDA to the growth medium or in conditions of Zincdepletion by addition of a Zinc chelator such as TPEN to the growthmedium such as MH agar) in the log phase of growth. TPEN can be used ata concentration of 10-30 uM, such as 20 uM, for example in MH agar. Thiscan be suspended in a medium with BSA (such as Hanks medium with 0.3%BSA) in order to obtain a working cell suspension adjusted toapproximately 20000 CFU/ml. A series of reaction mixes can be mademixing a series of two-fold dilutions of sera to be tested (suitablyheat-inactivated at 56° C. for 30 min) [for example in a 50 well volume]and the 20000 CFU/ml meningococcal strain suspension to be tested [forexample in a 25 well volume]. The reaction vials should be incubated(e.g. 37° C. for 15 minutes) and shaken (e.g. at 210 rpm). The finalreaction mixture [for example in a 100 μl volume] additionally containsa complement source [such as 25% final volume of pretested baby rabbitserum], and is incubated as above [e.g. 37° C. for 60 min]. A sterilepolystyrene U-bottom 96-well microtiter plate can be used for thisassay. A aliquot [e.g. 10 μl] can be taken from each well using amultichannel pipette, and dropped onto Mueller-Hinton agar plates(suitably containing 1% Isovitalex and 1% heat-inactivated Horse Serum)and incubated (for example for 18 hours at 37° C. in 5% C02). In oneaspect, individual colonies can be counted up to 80 CFU per aliquot. Thefollowing three test samples can be used as controls:buffer+bacteria+complement; buffer+bacteria+inactivated complement;serum+bacteria+inactivated complement. SBA titers can bestraightforwardly calculated using a program which processes the data togive a measurement of the dilution which corresponds to 50% of cellkilling by a regression calculation.

Alternatively, the synergistic response may be characterised by theefficacy of the combination of anigens in an adhesion blocking assay. Inone aspect the extent of blocking induced by antisera raised against thecombination of antigens is significantly improved compared with usingantisera raised against the antigens by themselves, particularly atsuboptimal doses of antibody.

The teaching of all references in the present application, includingpatent applications and granted patents, are herein fully incorporatedby reference. Any patent application to which this application claimspriority is incorporated by reference herein in its entirety in themanner described herein for publications and references.

For the avoidance of doubt the terms ‘comprising’, ‘comprise’ and‘comprises’ herein is intended by the inventors to be optionallysubstitutable with the terms ‘consisting of’, ‘consist of’, and‘consists of’, respectively, in every instance. As used in thisspecification and claim(s), the words “comprising” (and any form ofcomprising, such as “comprise” and “comprises”), “having” (and any formof having, such as “have” and “has”), “including” (and any form ofincluding, such as “includes” and “include”) or “containing” (and anyform of containing, such as “contains” and “contain”) are inclusive oropen-ended and do not exclude additional, unrecited elements or methodsteps.

Embodiments herein relating to “vaccine compositions” of the disclosureare also applicable to embodiments relating to “immunogeniccompositions” of the disclosure, and vice versa.

The term “about” (or “around”) in all numerical values allows for a 5%variation, i.e. a value of about 1.25% would mean from between1.19%-1.31%.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for themeasurement, the method being employed to determine the value, or thevariation that exists among the study subjects.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof is intended to include atleast one of: A, B, C, AB, AC, BC, or ABC, and if order is important ina particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

Reference to blebs, vesicles and outermembrane vesicles herein is alsointended to be a reference to all isolated membrane-derivedproteinaceous products known to persons of skill in the art such asblebs, microvesicles, OMVs, OMPC (outer membrane protein complex), ormembrane ghosts, and the like.

Reference to “for protection” and “for treatment” in all instancesherein can clearly alternatively be written “for use in the prevention”or “for use in the treatment”, respectively.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of thisdisclosure have been described in terms of suitable embodiments, it willbe apparent to those of skill in the art that variations may be appliedto the compositions and/or methods and in the steps or in the sequenceof steps of the method described herein without departing from theconcept, spirit and scope of the disclosure. All such similarsubstitutes and modifications apparent to those skilled in the art aredeemed to be within the spirit, scope and concept of the disclosure asdefined by the appended claims.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the disclosure.The principal features of this disclosure can be employed in variousembodiments without departing from the scope of the disclosure. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine study, numerous equivalents to the specific proceduresdescribed herein. Such equivalents are considered to be within the scopeof this disclosure and are covered by the claims. All publications andpatent applications mentioned in the specification are indicative of thelevel of skill of those skilled in the art to which this disclosurepertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

The disclosure will be further described by reference to the following,non-limiting, examples:

Example 1 L2 N. meningitidis strains express lower level of fHbp thanStrains Producing a LOS Containing a L3-Like Inner Core Methods AntigenPreparations

Recombinant fHBPs variants (v) A and B were produced in BL21 (DE3) E.coli after IPTG induction and purification using IMAC column. The fhbpsequences were derived from strains MC58 (fHBP B) and 2996 (fHBP A).Genes were cloned in pET24b plasmid without the nucleotide sequencecorresponding to the leader sequence and with a His-Tag in C-ter.Recombinant nadA was also produced in BL21 E. coli and the sequence wasderived from strain 2996.

Animal Procedures:

Groups of 300F1 mice were immunized three times by the intramuscular(IM) route on day 0, 21 and 28 with purified recombinant proteinsadsorbed onto Al(OH)₃. On day 42, blood samples were taken for serum.Mice sera were from experiments 20080232.

Western Blot with Whole Cell Preparations

N. meningitidis strains were cultivated overnight on MH agar plates at37° C. +5% CO₂. They were sub-cultured for 4 hours on MH agar plateswith 20 μM TPEN (zinc chelator) 37° C. +5% CO₂.

Inactivation was performed by incubating the cells harvested from agarplates in PBS-PMSF (200 μM)-azide (0.2%) ON at 37° C. Inactivated cellswere then washed in PBS and frozen at −20° C.

Ten microgram of cell preparations were loaded per wells and proteinswere separated under reducing condition using 12% gels (Novex), and thenthe proteins were transferred onto nitrocellulose membranes. Afterblocking non-specific binding sites, the membranes were incubated 2 hwith a mix of sera from mice immunized with either fHBP A, fHBP B orNadA. For Tdfl detection, the membranes were incubated either withanti-Tdfl rabbit serum or a mix of 4 anti-Tdfl Mabs. After washing,membranes were incubated with anti-mouse Ig biotinylated antibodies(Amersham). Binding of antibodies was detected usingstreptavidine-peroxidase conjugate (Amersham).

For each strain the level of expression of fHBP, Tdfl and NadA wasassessed by Western-Blot. Expression level was scored as followed: highlevel of expression, ++; intermediate expression level, +; lowexpression level, +/− and non-detectable expression, − as exemplified inFIG. 1.

Identification of fhbp Alleles:

For most strains, fHbp allele was determined by PCR typing as describedby Beerninck and collaborators in 2006. For some strains, the completefhbp locus was amplified from crude MenB lysate by PCR with primers. ThePCR fragment was then purified with High Pure PCR Purification Kit(Roche) and sequenced by the Sanger method. The sequence type (family Aor B) was deduced after comparison with family A and B referencesequences (2996 and MC58 respectively) using Lasergene MegAlign-ClustalXsoftware as described in Fletcher et al, 2004 (FIG. 2).

LOS Inner-Core Compositions

-   -   LOS inner-core compositions were deduced after analysis of        presence/functionality of Ipt3, Ipt6, lot3/oac1 and IgtG genes        with the following rules:        -   if lot3/oac1 is present and IN phase, GlcNAc is O-acetylated        -   if IgtG is present and IN phase, glucose linked on position            3 of HepII.        -   if Ipt6 is present, PEA linked on position 6 of HepII        -   if Ipt3 is present and IgtG absent or OUT phase            PEA linked on position 3 of HepII        -   if Ipt3 is present and IgtG IN phase            no PEA linked on position 3 of HepII        -   if Ipt3 is absent            no PEA linked on position 3 of HepII    -   The following nomenclature was used to characterized the        different inner-core structure:        -   L3=one PEA on position 3 of HepII with or without additional            Acetyl on inner core GlcNAc. Such inner-core is associated            with the following LOS immunotypes: L1, L3, L7, L8        -   L2=one PEA on position 6 and one glucose on position 3 of            HepII and one additional Acetyl on inner core GlcNAc        -   L3v=two PEA's on HepII (on positions 3 and 6) and in general            one additional Acetyl on inner core GlcNAc        -   L5=one glucose on position 3 of HepII and one additional            Acetyl on inner core GlcNAc        -   LX=no PEA or Glucose on position 3 and 6 of HepII and one            additional Acetyl on inner core GlcNAc

Results

-   -   The LOS inner core of 155 strains was typed by molecular method        (Table 1). These strains were        -   either selected randomly amongst disease isolates recently            isolated (after 2004) in UK (n=53), Germany (n=40) and Spain            (n=47).        -   or isolated before 2002 from patients in 5 different            countries (n=15).    -   Among the 140 recently isolates, the majority of strains produce        an L3 inner-core (from 66 to 94%). A L2 inner-core is observed        in 2 and 2.5% of English and German strains respectively while        it is found in more than 17% of Spanish strains.    -   Among the 155 clinical isolates, 52 strains were analysed for        fHBP and NadA gene occurrence, allele and fHBP, NadA, Tdfl        expression. These 52 were selected as followed: the 15 strains        isolated before 2002, 10 randomly selected recent isolates per        country (UK, Germany and Spain), and all recent L2 (Table 2).    -   The fhbp gene is present in all the 52 strains, 62% (32/52) of        strains possess the allele B and 38% (20/52) the allele A.    -   There are no apparent association between the inner-core LOS        type and fhbp allele as the fhbp B allele is detected in 68%        (21/31) and 60% (9/15) of L3 and L2 strains, respectively    -   There are no apparent association between the fHBP allele and        the fHBP expression since fHBP is well expressed (+ or ++) in        50% and 62% of strains possessing the fhbp A allele and the fhbp        B allele, respectively.    -   However, while only 19% (6/31) of L3 strains produce low or        undetectable level of fHBP, low/null expression of fHBP was        observed for 93% (14/15) of L2 strains.    -   Further it was found that many of the strains of L2 immunotype        were also of ST11 clonal complex.    -   The nadA gene is present in 44% of analyzed strains (23/52) but        only 25% of strains express detectable amount of NadA by Western        Blot (13/52).    -   In contrast to fHBP expression, there are no relation between        the LOS inner-core and the expression of NadA. Indeed, only 16%        of L3 and 27% of L2 strains produce detectable amount of NadA.    -   Among the 22 of the 52 strains that express no or low level of        fHBP, only 4 express detectable level of NadA.    -   The expression of Tdfl was analyzed using a mix of four        monoclonal antibodies directed against Tdfl and/or a polyclonal        serum from rabbit immunized with recombinant Tdfl. Among the 51        strains tested, 94% express detectable amount of Tdfl (48/51).    -   Among the 22 strains expressing low or undectable level of fHBP,        all but one produces Tdfl. This strain does not possess the nadA        gene.

Conclusion

The results suggest that L2 strains tend to produce significantly lowerlevel of fHBP than strain expressing a L3 inner-core LOS. Thisobservation argues against a MenB vaccine based only on fHBP, especiallyin countries such as Spain where L2 strains represent 17% of recentclinical isolates. In addition, all the Spanish L2 strains tested do notexpress detectable amount of NadA.

Among the 22 strains that express no or low level of fHBP, only 4express detectable level of NadA while all but one express Tdfl.

TABLE 1 Inner-core typing of the 155 invasive menB strains (including140 recently isolated) L3 (%) L2 (%) L3V (%) other (%) Germany (n = 40)85 2.5 5 7.5 UK (n = 53) 94 2 0 4 Spain (n = 47) 66 17 13 4 All (n =140) 82 7 6 5 Others (n = 15) 60 33 7 0

TABLE 2 Characteristics of the 52 strains analyzed: LOS inner-core type,fhpb allele (gene), level of expression of fHBP, Presence of nadA gene,level of expression of NadA, expression of TdfI (using MAbs and/orrabbit serum). LOS (inner- fHBP fHBP NadA NadA Tdfl WB Strains Countrycore typing) (gene) WB (gene) WB (MAbs/rabbit PAb) M97-250687 UK L3 B++ + + + M01-240013 UK L3 A + − − + M01-240101 UK L3 B + − − +M01-240149 UK L3 B + − − + M01-240185 UK L3 B + + − NT M01-240355 UK L3A + − − + M05-0240072 UK L2 B − + − + M05-0240210 UK L3 B + − − +M05-0240471 UK L3 B +/− − − + M05-0240697 UK L3 B + − − + M05-0241043 UKL3 B + − − + M05-0241255 UK L3 A + − − + M06-0240116 UK L3 B + − − +M06-0240359 UK L3 A − − − + M06-0240707 UK L3 A − − − − M06-0240928 UKL3 A + − − + DE10038_05 Germany L3 B + − − + DE10250_05 Germany L3 B + −− + DE10302_05 Germany  L3v A + + +/− − DE10410_05 Germany LX A − + − +DE10427_05 Germany L2 A + + +++ − DE10461_06 Germany L5 B ++ + + +DE10523_06 Germany L3 B + − − + DE10561_06 Germany L3 B + − − +DE10620_06 Germany L3 A +/− − − + DE10674_06 Germany L3 A + − − +DE10690_06 Germany L3 B +/− − − + DE10772_06 Germany L3 B + + + + 17540Spain L2 B − + − + 17607 Spain L3 B ++ + ++ + 17639 Spain L2 B +/− + − +17662 Spain  L3v A + − − + 17710 Spain L3 A ++ − − + 17763 Spain L2 B+/− + − + 17787 Spain L2 B +/− + − + 17810 Spain L3 A + − − + 17908Spain L2 B +/− + − + 17938 Spain L3 B ++ + ++ + 17981 Spain L2 B +/− −− + 18025 Spain L5 B ++ + +/− + 18064 Spain L3 B ++ + + + 18082 Spain L2B +/− + − + 18116 Spain L2 B +/− + − + BZ232 The Netherlands L2 A +/− −− + 760676 The Netherlands L2 A − + + + H44/76 Norway L3 B ++ − − +N98/254 NZ L3 B + − − + NZ124 NZ L3 B +/− − − + 2986 US? L2 A − + ++ +3356 US? L2 A − − − + 6275 US  L3v A − or +/− + ++ + B16B6 US L2 A − ++/− +

Example 2 Improvement of the Efficacy of fHbp Based Vaccine by Additionof Tdfl Methods Antigen Preparations

Recombinant fHBPs variants (v) A and B were produced in BL21 (DE3) E.coli after IPTG induction and purification using IMAC column. The fHbpsequences were derived from strains MC58 (fHBP B) and 2996 (fHBP A).Genes were cloned in pET24b plasmid without the nucleotide sequencecorresponding to the leader sequence and with a His-Tag in C-ter.

Animal Procedures:

Groups of 10-30 mice were immunized three times by the intramuscular(1M) route on day 0, 21 and 28. Each injection contained either OMVantigen normalized to 5 ug of protein and formulated with AS04 AdjuvantSystem (AIP04 plus 3-O-desacyl-4′ monophosphoryl lipid A) or withmonovalent fHBP vaccine (fHBP A or fHBP B) adsorbed onto Al(OH) orbivalent fHbpA+B vaccine. On day 42, blood samples were taken for serum.Mice sera were from experiments 20040652, 20070371 and 20080083.

Western Blot with Whole Cell Preparations

N. meningitidis strains were cultivated overnight on MH agar plates at37° C. +5% CO2. They were subcultured for 4 hours on MH agar plates with20 μM TPEN (zinc chelator) 37° C.+5% CO2. Inactivation was performed byincubating the cells harvested from agar plates in PBS-PM SF (200μM)-azide (0.2%) ON at 37° C. Inactivated cells were then washed in PBSand frozen at −20° C.

Ten microgram of cell preparations were loaded per wells and proteinswere separated under reducing condition using 12% gels (Novex), and thenthe proteins were transferred onto nitrocellulose membranes. Afterblocking non-specific binding sites, the membranes were incubated 2 hwith a monoclonal antibodiy directed against Tdfl or with a mix of serafrom mice immunized with either fHBP A or fHBP B sera. After washing,membranes were incubated with anti-mouse Ig biotinylated antibodies(Amersham). Binding of antibodies was detected usingstreptavidine-peroxidase conjugate (Amersham).

For each strain the level of expression of fHBP was assessed byWestern-Blot. Expression level was scored as followed: high level ofexpression, ++; intermediate expression level, +; low expression level,+/−; non-detectable expression, −.

SBA

N. meningitidis strains were cultivated overnight on Petri Dishes at 37°C. +5% CO. They were sub-cultured for 4 hours on Petri Dishes without orwith 20 μM TPEN (zinc chelator) 37° C. +5% CO2. Serum samples (pooledsera) were inactivated for 40 min at 56° C. and then diluted 1110 or1150 in PBS-glucose 0.1% and then twofold diluted in a volume of 25 μLin flat bottom microplates. Then 25 μL of a mix of bacteria (diluted inPBS-glucose 0.1% to yield—100-150 CFU per well) and baby rabbitcomplement (final concentration in microwell: 12.5% v/v) was added tothe serum dilution. After 75 min of incubation at 37° C. under shaking,2 layers of agar (0.9%) were added to the wells. The microplates wereincubated overnight at 35 or 37° C. +CO2. The CFU's were counted and thepercentage of killing was calculated. The SBA titer is the dilutiongiving 50% of killing.

Identification of fhbp Alleles:

The complete fhbp locus was amplified from crude MenB lysate by PCR withprimers. The PCR fragment was then purified with High Pure PCRPurification Kit (Roche) and sequenced by the Sanger method. Thesequence type (variant 1, 2 or 3) was deduced after comparison withvariant 1, 2 and 3 references sequences using LasergeneMegAlign-ClustalX software.

Results

Different N. meningitides strains were tested in SBA using sera frommice immunized with monovalent fHbp vaccine (fHbp A or fHbp B) or abivalent fHbp vaccine (fHbp A+B). These strains were selected based onthe expression level of fHbp determined by Western blot and by theirfHbp allele. The results (Table 3) clearly indicate that fHbp is notable to induce a cross-protective fHbp response since sera from miceimmunized with fHbp B are not bactericidal against strains producing afHbp A proteins (and the reverse was also observed). Sera from miceimmunized with fHBPA and fHBP B elicited the production of bactericidalantibodies capable to mediate the complement killing of strainsexpressing either the fHbp A or the fHbp B protein. In addition, thelevel of expression of fHbp by the targeted SBA strain impact also onthe bactericidal titers: lower is the fHbp expression level lower arethe bactericidal titers. These results illustrate the need to improve avaccine based on fHbp by adding other antigens in order to protectagainst strains producing low or non-detectable level of fHbp.

TABLE 3 Serum bactericidal titers of anti-fHBP mice sera against a panelof N meningitidis strains expressing different fHBP proteins atdifferent level. H44/76 NZ98/124 608B S3446 760676 fHbp B (++) fHbp B(+/−) fHbpB (−) fHbp A (+/−) fHbpA (−) Ctrl sera <50 <50 <50 <50 <50anti-fHbp (B) sera >2560 309 <50 <50 <50 anti-fHbp (A) sera 125 60 <50396 <50 anti-fHbp (A&B) sera >2560 221 <50 1104 <50

Mice were immunized with different OMV preparations obtained fromrecombinant H44/76 strains having a common background: porA KO, galE LOSand capsule minus. The different preparations are differentiated by thelevel of Tdfl and fHbp. Control OMVs (Ctrl OMVs) had no detectableamount of Tdfl and fHbp. Tdfl OMVs were produced from a strain thatoverexpressed Tdfl and Tdfl-fHBPOMVs displayed both Tdfl and fHbp. Thesera were analysed in SBA using a panel of H44/76 strains expressingdifferent level of Tdfl. The wild type (WT) strain did not express anydetectable amount of Tdfl while a recombinant H44/76 strain transformedwith the pfP10 plasmid containing the Tdfl gene under the pTac promoterproduced high level of Tdfl in presence of ITPG (IPTG) (Table 4).

The sera from mice immunized with the control preparation (Ctrl-OMVs)were not bactericidal. Only the strain expressing high amount of Tdfl(IPTG) was killed by anti-Tdfl antibodies in presence of complement. Bycontrast, sera from mice immunized with Tdfl-fHbp OMV preparationmediated the complement killing of the two H44/76 strains, via thepresence of anti-fHbp antibodies and as observed there was a correlationbetween the bactericidal titer and the level of Tdfl produced by thetargeted strains (Table 4).

TABLE 4 Serum bactericidal titers on different H44176 strains producingdifferent level of Tdfl (wild type, WT; or overproducing Tdfl via IPTGinduction; IPTG). SBA titers WT IPTG anti-Ctrl OMV sera 50 50 anti-TdflOMV sera 50 679 anti-Tdfl-fHbp OMV sera 592 1146

Example 3 Improvement of the Efficacy of fHbp Based Vaccine by Additionof Tdfl Methods

Antigen Preparations

Outer membranes vesicles (OMVs) were produced using classical 0.5% DOCextraction from different recombinant H44/76 strains (porA KO, capsuleminus, galE LOS and over-producing different outer-membrane proteins).

Recombinant fHBPs variants (v) A and B were produced in BL21 (DE3) E.coli after IPTG induction and purification using IMAC column. The fhbpsequences were derived from strains MC58 (fHBP B) and 2996 (fHBP A).Genes were cloned in pET24b plasmid without the nucleotide sequencecorresponding to the leader sequence and with a His-Tag in C-ter.

Animal Procedures:

Groups of 10-30 mice were immunized three times by the intramuscular(IM) route on day 0, 21 and 28. Each injection contained 5 μg ofmonovalent fHBP vaccine (fHBP A or fHBP B) adsorbed onto Al(OH)₃. On day42, blood samples were taken for serum. Mice sera were from experiments20080790 and 20090265.

Groups of 10 guinea-pigs were immunized three times by the intramuscular(IM) route on day 0, 14 and 28. Each injection contained either OMVantigen normalized to 10 μg of protein and formulated with AIPO₄. On day42, blood samples were taken for serum. Guinea pig sera were fromexperiments 20090266.

SBA

N. meningitidis strains were cultivated overnight on Petri Dishes at 37°C. +5% CO₂. They were sub-cultured for 4 hours on Petri Dishes withoutor with 20 μM TPEN (zinc chelator) 37° C. +5% CO₂. Serum samples (pooledsera) were inactivated for 40 min at 56° C. and then diluted 1/10 or1/50 in PBS-glucose 0.1% and then twofold diluted in a volume of 25 μlin flat bottom microplates. Then 25 μl of a mix of bacteria, fromagar-plate culture or after cell contact (diluted in PBS-glucose 0.1% toyield ˜100-150 CFU per well) and baby rabbit complement (finalconcentration in microwell: 12.5% v/v) was added to the serum dilution.After 75 min of incubation at 37° C. under shaking, 2 layers of agar(0.9%) were added to the wells. The microplates were incubated overnightat 35 or 37° C. +CO₂. The CFU's were counted and the percentage ofkilling was calculated. The SBA titer is the dilution giving 50% ofkilling.

Results

Anti-fHbpB sera were tested in SBA with strains expressing the fHbpfamily B, while anti-fHbpA sera were used in SBA against strainsexpressing the fHbp from family A. Sera from guinea-pigs immunized withOMVs (blebs) produced from a strain over-expressing Tdfl were testedagainst both fHbpB and fHbpA strains.

Anti-Tdfl and anti-fHbp sera were tested alone or were mixed before toperform SBA in presence or absence of TPEN.

A panel of 16 strains was used in SBA. Among those, 5 to 7 were killedby anti-fHbp antibodies (pending the absence or presence of TPEN in SBAculture condition) (SBA titer >128). In absence of TPEN, only 3 strainswere killed by sera from guinea-pig immunized with Tdfl-blebs while inpresence of TPEN, 12 strains were killed.

When anti-fHbp and anti-Tdfl blebs sera are mixed, there is a generaltrend to improve the SBA titers, showing at least an additive impact ofanti-fHBp and anti-Tdfl blebs serum bactericidal activity.

TABLE 5 Strain DE- H44/ NZ98/ M01- 10690- M01- M01- M01- M05- M05- M98-76 MC58 254 240101 06 240149 18025 240013 240355 0240471 760676 175400240072 250771 Serogroup B B B B B B B B B B B B B B Immuntype L3 L3 L3L3 L3 L2 L3 L3 L3 L3 L2 L2 L2 L2 fHbp family B B B B B B B A A B A B B AfHbp ++ ?? + + +/− ?? +/− + + +/− − − − ?? expression SBA with TPENAnti-Tdfl 599 1460 1367 1449 656 2049 585 1650 1577 1200 50 504 50 1215blebs sera Anti fHbp 3539 5786 388 509 169 2104 4373 50 50 50 50 50 5050 sera Mix of 5941 7264 1284 1690 1127 4782 8406 2741 2540 308 50 50550 1292 both sera SBA without TPEN Anti-Tdfl 220 160 50 50 50 50 161 5050 50 50 50 118 50 blebs sera Anti fHbp 3383 3993 110 494 50 2293 380450 50 50 50 50 50 50 sera Mix of 3104 5144 50 486 50 3645 5432 50 50 5050 50 50 50 both sera

Example 4

Protective effects of different vaccine compositions in SBA against 19N. meningitidis strains using baby rabbit serum as complement source.Results are expressed as the percentage of strains killed (titers≧1/128). Comparison of strain coverage induced by the bivalent 15% LOSOMV vaccine, the bivalent fHBP and the pentavalent subunit vaccinecomprising-fHbp (GNA1870) NadA, GNA2132 (Lipo28), GNA1030, GNA2091.

TABLE 6 Number (%) of strains killed (SBA ≧ 1/128) Bivalent BivalentPentavalent LOS type N^(a) Serogroup^(b) OMVs fHBP vaccine L2 6 B, W  4(67%) 0  1 (17%) L3,7 4 B  3 (75%) 4 (100%)  3 (75%) L3v 6 B, C, W, Y  6(100%) 2 (33%)  6 (100%) L4 1 C  0 1 (100%)  0 L10 1 A  0 0  0 L11 1 A 1 (100%) 0  1 (100%) All 19 14 (74%) 7 (37%) 11 (58%) ^(a)Number ofstrains tested expressing the respective LOS type ^(b)Serogrouprepresented by the strains tested in SBA for respective LOS type

Same results but presented in more detail:

TABLE 7 L3 L3V L3V and L3orL4 B B B B B W Y B B C H44/76 M667 NZ124S3446 6275 3193 S1975 2991 608B C11 fHbpexpression(WB) ++ ++ + + − NTNT + − NT L7 + L2 non-ads 3292 1611 132 54 7122 12734 25600 9738 256001023 L7 + L2 ads 1164 747 158 10 3327 13054 9840 25600 6326 556 T

3 +

2 non-ads 40960 2542 504 112 5985 18153 25600 25600 9371 1105 T

3 +

2 ads 2705 1517 343 61 747 4879 9728 25600 3460 463 Protein9

15 11 10 10 10 10 546 10 10 50 Protein9

15 10 10 10 10 10 567 10 10 50 GNA2

18 10 57 102 10 10 477 113 10 50 GNA

1942 416 33 10 10 10 10 55 10 50 N

13 260 10 10 118 5144 7495 >20480 908 849 5

B 3759 3737 104 319 901 4607 12780 >20480 635 506 fHbpB 5633 11354 60435 <50 <50 <50 <50 <50 <50 fHbpA 125 120 232 396 <50 <50 <50 153 <50 <50fHbpA + B 8991 5119 741 1104 <50 <50 123 1466 <50 189 GNA1870WesternBlot++ ++ + + − NT NT + − NT L4? L2 L10 L11 C B B B B B W A A C19 760676B16B6 2986 3356 BZ232 S4383 3125 F8238 fHbpexpression(WB) NT − − − − −NT NT NT L7 + L2 non-ads 10 3050 1148 7328 22 10 1041 10 4240 L7 + L2ads 10 1192 715 4542 87 10 935 10 3221 T

3 +

2 non-ads 10 2450 415 3929 34 10 2164 22 4348 T

3 +

2 ads 10 406 281 2088 73 10 369 10 4073 Protein9

10 10 10 10 10 10 23 10 10 Protein9

10 10 10 10 10 10 10 10 10 GNA2

266 10 10 10 29 10 10 10 10 GNA

10 10 10 10 10 10 23 10 10 N

10 13 251 997 10 10 10 10 3137 5

B 53 11 108 1013 59 10 10 10 428

fHbpB <10 <50 <50 <50 <10 <10 <50 <10 <50 fHbpA 64 <50 <50 <50 <10 <10<50 <10 <50 fHbpA + B 513 <50 <50 <50 <10 101 <50 <10 <50GNA1870WesternBlot NT − − − − − NT NT NT

indicates data missing or illegible when filed

Example 5 Manufacture of Fusion Proteins

1. Different Fusion Proteins from fHbp were Expressed in E. coliStrains.

TABLE 8 Concen- tration Volume Quantity LVL ID description (mg/ml) (ml)Buffer (mg) LVL489 Fhbp from Family 0.30 15 PBS 4.5 A (full-length - 1XSEQ ID NOS. 9 and 10) LVL490 Fhbp from Family 0.44 10 PBS 4.4 B(full-length - 1X SEQ ID NOS. 11 and 12) LVL491 Fusion w/o 0.50 10 PBS5.0 mutation (wild 1X type sequence - SEQ ID NOS. 16 and 17)) LVL511Fusion protein A - 0.59 10 PBS 5.9 SEQ ID NOS. 1X 18 and 19 LVL512Fusion protein B - 0.56 10 PBS 5.6 SEQ ID NOS. 1X 20 and 21 LVL513Fusion protein C - 0.25 15 PBS 3.8 SEQ ID NOS. 1X 22 and 23 LVL514Fusion protein E - 0.44 10 PBS 4.4 SEQ ID NOS. 1X 24 and 25

2. Host Strain:

T7 Express competent E. coli (NEB catalogue number C2566H): EnhancedBL21 derivative. T7 RNA Polymerase in the lac operon—no λ prophage.Deficient in proteases Lon and OmpT. Resistant to phage T1 (fhuA2). Doesnot restrict methylated DNA (McrA⁻, McrBC⁻, EcoBr⁻m⁻, Mrr⁻). B Strain.Free of animal products. Genotype: fhuA2 lacZ::T7 gene1 [Ion] ompT galsulA11 R(mcr-73::miniTn10—Tet^(S))2 [dcm] R(zgb-210::Tn10—Tet^(S)) endA1Δ(mcrC-nmr)114::IS10:

3. Recombinant Proteins: (Numbering Given in Respect of Full LengthSequence)

The Family B part of the fusion exemplified herein starts from aminoacid position 73 of the full length MC58 sequence, which full lengthsequence itself comprises a mature sequence starting at amino acid 66.(The 7 N-ter aa (CSSGGGG—SEQ ID NO. 13) are replaced by MHHHHHH—SEQ IDNO. 14 to allow purification).

MC58 part of the fusion finishes at residue 200 ( . . . DIA) if we takeaccount the numbering of the full length MC58 protein sequence, withresidue 200 of the full length sequence corresponds to the residue 135of the mature MC58 sequence.

The 8047 part of the fusion exemplified herein begins at residue 155 ofthe full length (273 amino acid) 8047 protein sequence (GEH . . . ). Thepeptide GENT (SEQ ID NO. 15) is identical in family A and B, thejunction between the 2 parts is the Gly residue. Residue 155 of the fulllength sequence corresponds to the residue 136 of the mature sequence.

TABLE 9 Recombinant plasmids ID N-terminal C-Terminal* LVL489 MHH FamA(Strain 8047/A.A.: 27 to 273) HHH H 1  7 8 254 LVL490 MHH FamB (StrainMC58/A.A.: 73 to 320) HHH H 1  7 8 255 LVL491 MHH FamB (StrainMC58/A.A.: 73 FamA (Strain 8047/A.A.: HHH to 200) 155 to 273) H 1  7 8135 136 254 LVL511 MHH FamB (Strain MC58/A.A.: 73 FamA (Strain8047/A.A.: HHH to 200) 155 to 273) H E217A, T238A**: Disruption offactor H- binding 1  7 8 135 136 254 LVL512 MHH FamB (Strain MC58/A.A.:73 FamA (Strain 8047/A.A.: HHH to 200) 155 to 273) H E217A**: Disruptionof factor H-binding 1  7 8 135 136 254 LVL513 MHH FamB (StrainMC58/A.A.: 73 FamA (Strain 8047/A.A.: HHH to 200) 155 to 273) H E217A,T238A**: Disruption of factor H-binding D146E, K148GR and S203R**: Torestore the family B MAb502 1  7 8 135 135 255 LVL514 MHH FamB (StrainMC58/A.A.: 73 FamA (Strain 8047/A.A.: HHH to 200) 155 to 273) H E217A,T238A**: Disruption of factor H-binding D146E, K148GR and S203R**: Torestore the family B MAb502 R229K**: Could help to restore the Mab5021  7 8 135 136 255

4. Expression of the Recombinant Proteins:

4.1—Transformation

Transformation of Escherichia coli T7 Express with plasmid DNA wascarried out by standard methods with CaCl₂-treated cells (Hanahan D.<<Plasmid transformation by Simanis.>> In Glover, D. M. (Ed), DNAcloning. IRL Press London. (1985): p. 109-135.).

Recombinant plasmids ID Host strain Plate agar LVL489, LVL490, LVL491,LVL511, T7 LB + agar- LVL512, LVL513 and LVL514 Express^(A) 1.5%^(B) 40μg/ml kanamycin^(c) ^(A)NEB (catalogue number C2566H) ^(B)BBL ™ SelectAPS ™ LB broth base, BD, MD, USA (catalogue number: 292438); Agar,Laboratoire MAT, QC, Canada (catalogue number: AP-0108) ^(C)Sigma, ON,Canada (catalogue number: K-4000)

4.2—Culture

Confluent agar plate inoculated with Escherichia coli T7 Express+plasmidfrom transformation (section 5.1) was stripped, ressuspended in culturemedia and used to inoculate 800 ml of LB broth (BD) ±1% (w/v) glucose(Laboratoire MAT, catalogue number: GR-0101)+antibiotic (as described insection 5.1) to obtain O.D._(600 nm) between 0.1 and 0.2.

Cultures were incubated at 37° C., 250 RPM until an O.D._(600 nm) around0.8. At this time, 1 ml of each culture was collected, centrifuged at 14000 RPM for 5 minutes and supernatants/pellets were frozen at −20° C.separately.

4.3—Induction

At O.D._(600 nm) around 0.8, the cultures T7 Express were cooled down(−20° C., 20 minutes) before inducing the expression of the recombinantprotein by addition of 1 mM isopropyl 6-D-1-thiogalactopyranoside (IPTG;EMD Chemicals Inc., catalogue number: 5815) and incubated overnight at22° C., 250 RPM.

4.4—Preparation of Samples after Induction

After the overnight induction (around 16 hours), O.D._(600 nm) wasevaluated after induction and culture was centrifuged at 14 000 RPM for5 minutes and supernatant/pellets were frozen at −20° C. separately.

5. Purification:

Bacterial pellet was resuspended in 20 mM bicine buffer (pH 8.0)containing 500 mM NaCl and a mixture of protease inhibitor (Complete,Roche). Bacteria were lysed using a Constant System 1.1 KW 2×30 000 PSI.Soluble (supernatant) and insoluble (pellet) components were separatedby centrifugation at 20 000 g for 20 min at 4° C.

The 6-His tagged-protein was purified under native conditions on IMACusing Profinia standard protocol (flow rate: 2 ml/min). The solublecomponents were loaded on a 5 ml H is Trap column (BioRad)preequilibrated with the same buffer used to bacterial resuspension.After loading on the column, the column was washed with the same buffer.We used 20 mM bicine buffer (pH 8.0) containing 500 mM NaCl, 10 mMimidazole for the second wash. Elution was performed using a 20 mMbicine buffer (pH 8.0) containing 500 mM NaCl and 250 mM imidazole.Proteins were dialysed using PBS 1×pH 7.4 and dosing was determinedusing DC Protein Assay of BioRad.

SDS-Page:

Gel: NuPAGE 4-12% Bis-Tris Gel 1.0 mm×15 wells (Invitrogen catalognumber: NP0323BOX)

Preparations of samples, buffers and migration conditions were doneunder conditions recommended by the suppliers (Invitrogen).

Western Blot:

Preparations of buffer and migration conditions were done underconditions recommended by the suppliers (Invitrogen).

Membranes were blocked for 30 minutes at 37° C., 60 RPM using 3%milk/PBS 1× fresh solution. After the blocking incubation, PrimaryAntibodies were added consisting to α-6×His Tag (AbCam, cataloguenumber: ab9108-100) or α-fHbpA (200802032 pool g2, 18/06/08 D42) atdilution: 1:1000 or 1:400 respectively in 3% milk/PBS 1× fresh solutionfor 1 hour at 37° C., 60 RPM. After that, membranes were washed threetimes 5 minutes at room temperature using 0.02% Tween 20/PBS 1×.Secondary Antibodies were added using a goat anti-rabbit alkalinephosphatase (Jackson laboratory, catalogue number: 111-055-144) atdilution 1:14 000 or a Goat alkaline phoaphatase anti-IgG +IgM (H+L)mouse (Jackson laboratory, catalogue number: 115-055-068)) at dilution1:6 000 in 3% milk/PBS 1× fresh solution. Membranes were incubated for 1hour at 37° C., 60 RPM. After that, membranes were washed three times 5minutes at room temperature using 0.02% Tween20/PBS 1× before themembrane expositions to Alkaline Phosphatase substrate (Sigma FastNBT/BCIP, catalogue number:B5655-25TAB) under conditions recommended bythe suppliers.

Example 6 Fusion Protein A

This fusion comprises a part of the family B MC58 mature proteinsequence (from residue 1 to residue 135) and a part of the family A 8047mature protein sequence (from residue 136 to residue 254). In thisfusion, 2 residues (Glu217 and Thr238), identified by M. C. Schneider etal. as involved in the factor H-binding function of the protein, aremutated in Alanine.

These mutations in the nucleic sequence:

-   -   Codon GAA (nT 649, Glu217) is mutated in codon GCA (Ala217, *)    -   Codon ACC (nT 712, Thr238) is mutated in codon GCC (Ala238; *)

Sequence of this fusion—SEQ ID No. 18 (length: 254 aa):

  1 MHHHHHH VAA DIGAGLADAL TAPLDHKDKG LQSLTLDQSV RKNEKLKLAA  51QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QIEVDGQLIT LESGEFQVYK 101QSHSALTAFQ TEQIQDSEHS GKMVAKRQFR IGDIAGEHTA FNQLPDGKAE 151YHGKAFSSDD AGGKLTYTID FAAKQGHGKI EHLKTPEQNV ELAAAELKAD                 *                      * 201EKSHAVILGD TRYGSEAKGT YHLALFGDRA QEIAGSAAVK IGEKVHEIGI 251 AGKQ

The underlined sequence is the family B sequence coming from strainMC58. The other part of the fusion is the family A sequence coming fromstrain 8047.

Corresponding nucleic sequence (SEQ ID No. 19):

ATGCATCATCATCACCATCATGTTGCAGCAGATATTGGCGCAGGTCTGGCAGATGCACTGACCGCTCCGCTGGATCATAAAGATAAAGGTCTGCAGAGCCTGACCCTGGATCAGAGCGTTCGCAAAAATGAAAAACTGAAACTGGCAGCACAGGGTGCAGAAAAAACCTATGGTAATGGCGATAGCCTGAATACCGGCAAACTGAAAAATGATAAAGTGAGCCGCTTTGATTTTATTCGCCAGATTGAAGTTGATGGTCAGCTGATTACCCTGGAAAGCGGTGAATTTCAGGTGTATAAACAGAGCCATAGCGCACTGACCGCCTTTCAGACCGAACAAATTCAGGATAGCGAACATAGCGGTAAAATGGTTGCCAAACGCCAGTTTCGTATTGGTGATATTGCCGGTGAACATACCGCATTTAATCAGCTGCCGGATGGTAAAGCAGAATATCATGGCAAAGCCTTTAGCTCTGATGATGCCGGTGGTAAACTGACCTATACCATTGATTTTGCAGCCAAACAGGGTCATGGCAAAATTGAACATCTGAAAACACCGGAACAGAATGTTGAACTGGCAGCAGCAGAACTGAAAGCAGATGAAAAAAGCCATGCCGTTATTCTGGGTGATACCCGTTATGGTAGCGAAGCAAAAGGCACCTATCATCTGGCACTGTTTGGTGATCGTGCACAGGAAATTGCAGGTAGCGCAGCAGTTAAAATTGGCGAAAAAGTGCATGAAATTGGCATT GCCGGTAAACAG

Example 7 Fusion Protein B SEQ ID NOS. 20 and 21

Given that the Glu238 is not conserved in all strains that can bind thefactor H, this residue is potentially not critical for this binding.Thus a fusion protein B is proposed.

This fusion is based on fusion A in which only the amino acid Glu217(conserved in all analysed strains and very probably involved in thefactor H-binding) is mutated in Ala217.

Example 8 Fusion Protein C

This fusion based on fusion A, further including some mutations wereintroduced to restore the family B MAb502 epitope that is lost infusions A and B.

The residues Glu146→Arg149 and Arg204 of family B mature proteinsequence were identified as key residues for MAb502 recognition.

The residue Gly147 is already present in the fHbp family A matureprotein sequence. The amino acids Asp146, Lys148 and Ser203 of fHbpfamily A protein sequence are replaced by Glu146, Arg149 and Arg204,respectively. Moreover, a Glycine is introduced at position 147.

The mutations in the nucleic sequence:

-   -   Asp146→Glu146: GAC→GAA (nT 436)    -   Addition of Gly147: GGC (nT 439)    -   Lys148→Arg149: AAA→AGG (nT 445)    -   Ser203→Arg204: TCA→CGT (nT 610)

Sequence of the fusion (length: 255 aa) (SEQ ID No. 22):

  1 MHHHHHH VAA DIGAGLADAL TAPLDHKDKG LQSLTLDQSV RKNEKLKLAA  51QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QIEVDGQLIT LESGEFQVYK 101QSHSALTAFQ TEQIQDSEHS GKMVAKRQFR IGDIAGEHTA FNQLPEGGRA 151EYHGKAFSSD DAGGKLTYTI DFAAKQGHGK IEHLKTPEQN VELAAAELKA 201DEKRHAVILG DTRYGSEAKG TYHLALFGDR AQEIAGSAAV KIGEKVHEIG 251 IAGKQ

Corresponding nucleic sequence (SEQ ID No. 23):

ATGCATCATCATCACCATCATGTTGCAGCAGATATTGGCGCAGGTCTGGCAGATGCACTGACCGCTCCGCTGGATCATAAAGATAAAGGTCTGCAGAGCCTGACCCTGGATCAGAGCGTTCGCAAAAATGAAAAACTGAAACTGGCAGCACAGGGTGCAGAAAAAACCTATGGTAATGGCGATAGCCTGAATACCGGCAAACTGAAAAATGATAAAGTGAGCCGCTTTGATTTTATTCGCCAGATTGAAGTTGATGGTCAGCTGATTACCCTGGAAAGCGGTGAATTTCAGGTGTATAAACAGAGCCATAGCGCACTGACCGCCTTTCAGACCGAACAAATTCAGGATAGCGAACATAGCGGTAAAATGGTTGCCAAACGCCAGTTTCGTATTGGTGATATTGCCGGTGAACATACCGCATTTAATCAGCTGCCGGAAGGTGGTCGTGCAGAATATCATGGCAAAGCCTTTAGCTCTGATGATGCCGGTGGTAAACTGACCTATACCATTGATTTTGCAGCCAAACAGGGTCATGGCAAAATTGAACATCTGAAAACACCGGAACAGAATGTTGAACTGGCAGCAGCAGAACTGAAAGCAGATGAAAAACGTCATGCCGTTATTCTGGGTGATACCCGTTATGGTAGCGAAGCAAAAGGCACCTATCATCTGGCACTGTTTGGTGATCGCGCACAGGAAATTGCAGGTAGCGCAGCAGTTAAAATTGGCGAAAAAGTGCATGAAATTGGC ATTGCCGGTAAACAG

Example 9 Proposed Fusion Protein D

Fusion protein D Is based on fusion C in which only the amino acidGlu218, involved in the factor H-binding, is mutated in Ala218.

Example 10 Fusion Protein E

This fusion is based in fusion protein C. Some additional residues wereidentified as potentially involved in the MAb502 recognition. There arePro145, Phe227, Gly228, Lys230 and Glu233 in the family B mature proteinsequence.

To test the role of these residues, the fusion E was proposed, it is thefusion C in which these residues were inserted.

To restore all these potentially interesting residues in the fusion,only one codon mutation must be done on the fusion protein C construct:Arg230 in the family A mature protein sequence (strain 8047) must bemutated in Lys230.

Sequence of the fusion—SEQ ID no. 24 (length: 255 aa):

  1 MHHHHHH VAA DIGAGLADAL TAPLDHKDKG LQSLTLDQSV RKNEKLKLAA  51QGAEKTYGNG DSLNTGKLKN DKVSRFDFIR QIEVDGQLIT LESGEFQVYK 101QSHSALTAFQ TEQIQDSEHS GKMVAKRQFR IGDIAGEHTA FNQLPEGGRA 151EYHGKAFSSD DAGGKLTYTI DFAAKQGHGK IEHLKTPEQN VELAAAELKA 201DEKRHAVILG DTRYGSEAKG TYHLALFGD K  AQEIAGSAAV KIGEKVHEIG 251 IAGKQ

The mutations in the nucleic sequence:

-   -   Arg230→Lys230: CGC→AAA (nT 688)

Corresponding nucleic sequence (SEQ ID No. 25):

ATGCATCATCATCACCATCATGTTGCAGCAGATATTGGCGCAGGTCTGGCAGATGCACTGACCGCTCCGCTGGATCATAAAGATAAAGGTCTGCAGAGCCTGACCCTGGATCAGAGCGTTCGCAAAAATGAAAAACTGAAACTGGCAGCACAGGGTGCAGAAAAAACCTATGGTAATGGCGATAGCCTGAATACCGGCAAACTGAAAAATGATAAAGTGAGCCGCTTTGATTTTATTCGCCAGATTGAAGTTGATGGTCAGCTGATTACCCTGGAAAGCGGTGAATTTCAGGTGTATAAACAGAGCCATAGCGCACTGACCGCCTTTCAGACCGAACAAATTCAGGATAGCGAACATAGCGGTAAAATGGTTGCCAAACGCCAGTTTCGTATTGGTGATATTGCCGGTGAACATACCGCATTTAATCAGCTGCCGGAAGGTGGTCGTGCAGAATATCATGGCAAAGCCTTTAGCTCTGATGATGCCGGTGGTAAACTGACCTATACCATTGATTTTGCAGCCAAACAGGGTCATGGCAAAATTGAACATCTGAAAACACCGGAACAGAATGTTGAACTGGCAGCAGCAGAACTGAAAGCAGATGAAAAACGTCATGCCGTTATTCTGGGTGATACCCGTTATGGTAGCGAAGCAAAAGGCACCTATCATCTGGCACTGTTTGGTGATAAAGCACAGGAAATTGCAGGTAGCGCAGCAGTTAAAATTGGCGAAAAAGTGCATGAAATTGGC ATTGCCGGTAAACAG

Example 11 Proposed Fusion Protein F

It is based on fusion E in which only the amino acid Glu218, involved inthe factor H-binding, is mutated in Ala218.

Example 12 Effect of Antibodies Against fHbp on ST269 Clonal ComplexMethods

Antigen Preparations

Recombinant fHBPs variants (v) A and B were produced in BL21 (DE3) E.coli after IPTG induction and purification using IMAC column. The fhbpsequences were derived from strains MC58 (fHBP B) and 2996 (fHBP A).Genes were cloned in pET24b plasmid without the nucleotide sequencecorresponding to the leader sequence and with a His-Tag in C-ter.

Animal Procedures:

Groups of 10-30 mice were immunized three times by the intramuscular(IM) route on day 0, 21 and 28. Each injection contained 5 μg ofmonovalent fHBP vaccine (fHBP A or fHBP B) adsorbed onto Al(OH)₃. On day42, blood samples were taken for serum. Mice sera were from experiments20080790 and 20090265.

Groups of 10 guinea-pigs were immunized three times by the intramuscular(IM) route on day 0, 14 and 28. Each injection contained either OMVantigen normalized to 10 μg of protein and formulated with AIPO₄. On day42, blood samples were taken for serum. Guinea pig sera were fromexperiments 20090266.

SBA

N. meningitidis strains were cultivated overnight on Petri Dishes at 37°C. +5% CO₂. They were sub-cultured for 4 hours on Petri Dishes withoutor with 20 μM TPEN (zinc chelator) 37° C. +5% CO₂. Serum samples (pooledsera) were inactivated for 40 min at 56° C. and then diluted 1/10 or1/50 in PBS-glucose 0.1% and then twofold diluted in a volume of 25 μlin flat bottom microplates. Then 25 μl of a mix of bacteria, fromagar-plate culture or after cell contact (diluted in PBS-glucose 0.1% toyield ˜100-150 CFU per well) and baby rabbit complement (finalconcentration in microwell: 12.5% v/v) was added to the serum dilution.After 75 min of incubation at 37° C. under shaking, 2 layers of agar(0.9%) were added to the wells. The microplates were incubated overnightat 35 or 37° C. +CO₂. The CFU's were counted and the percentage ofkilling was calculated. The SBA titer is the dilution giving 50% ofkilling.

Two strains from the clonal complex ST269 were used: M01-240013 andM01-240101. Both strains were isolated in UK in 2001.

Results

Anti-fHbpB sera were tested in SBA with strain expressing the fHbpfamily B (M01-240101), while anti-fHbpA sera were used in SBA againststrain expressing the fHbp from family A (M01-240013). SBA wereperformed in absence or presence of TPEN.

Only one strain was killed by anti-fHbp antibodies (strain M01-240101)in presence of complement. The second strain, M01-240013 is not killedby anti-fHbp antibodies. As observed, bactericidal culture condition(absence or presence of TPEN) has no impact on SBA titers.

TABLE 1 bactericidal titer of anti-fHbp antibodies in presence of babyrabbit complement Strain M01-240101 M01-240013 fHbp family B A fHbpexpression + + SBA titer without TPEN Anti-fHbp B 494 Not testedAnti-fHbpA Not tested 50 SBA titer with TPEN Anti-fHbp B 509 Not testedAnti-fHbpA Not tested 50

Conclusion

Anti-fHbp antibodies were not able, alone, to provide effective killingof M01-240013 strain (from clonal complex ST269)

Example 13 Immunogenicity of Chimeric fHbp Proteins Methods

Antigen Preparations

Chimeric fHbp proteins with/without mutation of fH binding site wereprepared as described in the above examples (Examples 5-11).

Animal Procedures:

Groups of 20 mice were immunized three times by the intramuscular (IM)route on day 0, 21 and 35. Each injection contained 5 μg of chimericfHbp proteins adsorbed onto Al(OH)₃. On day 49, blood samples were takenfor serum. Mice sera were from experiment 20090833.

rSBA

N. meningitidis strains were cultivated overnight on Petri Dishes at 37°C. +5% CO₂. They were sub-cultured for 4 hours on Petri Dishes at 37° C.+5% CO₂. Serum samples (individual sera) were inactivated for 40 min at56° C. and then diluted 1/50 in PBS-glucose 0.1% and then twofolddiluted (8×) in a volume of 25 μl in flat bottom microplates. Then 25 μlof a mix of bacteria, from agar-plate culture (diluted in PBS-glucose0.1% to yield ˜50-250 CFU per well) and baby rabbit complement (finalconcentration in microwell: 12.5% v/v) was added to the serum dilution.After 75 min of incubation at 37° C. under shaking, 2 layers of agar(0.9%) were added to the wells. The microplates were incubated overnightat 33° C. +CO₂. The CFU's were counted and the percentage of killing wascalculated. The SBA titer is the dilution giving 50% of killing.

Individual sera were analysed and geometric mean titer was calculated.For geometric mean calculation, titer was set at 50 when killing forfirst dilution was below 50% killing or titer was set at 25600 ifkilling was higher than 50% at last dilution.

Results

Sera were tested in rSBA with strain expressing the fHbp family B(H44/76) or the fHbp family A (S3446) (table 1 below).

Immunisation with recombinant fHbpB induced the production ofbactericidal antibodies able to mediate the complement killing of H44/76strain (fHbp family B) but not S3446 strain (fHbp family A) (percentageof responders 100% and 10%, respectively). The recombinant fHbpA induceda low bactericidal antibody response againt the S3446 strain and a verylow response against the H44/76 strain.

Animals immunized with a chimeric fHbp (with or without fH binding sitemutation) were able to elicit a bactericidal response against bothH44/76 and S3446 strains (up to 100% and 90% responders against H44/76and S3446 respectively). The best response was achieved with theconstruction LVL-511 (two amino acid mutations).

TABLE 1 bactericidal titer of anti-chimeric fHbp antibodies in presenceof baby rabbit complement Chimaeric Chimaeric Chimaeric Chimaeric fHbpfHbp fHbp fHbp Chimaeric fH binding fH binding fH binding fH bindingWild type Wild type fHbp mutation A mutation B mutation C mutation Efhbp A fhbp B ctrl LVL-491 LVL-511 LVL-512 LVL-513 LVL-514 LVL-489LVL-490 (−) H44/76 GMT (50% killing) 846 2291 1017 762 1187 60 10121 58(fHbp family B) Responders (titer ≧100) 95%* 100% 100%* 100%* 100%* 25%*100%* 18%* S3446 GMT (50% killing) 301  466  190 114  112 93   60 50(fHbp family A) Responders (titer ≧100) 85%   90% 85% 55% 45% 45%  10%0% *n = 6 to 19 due to invalid results (low CFU)

Conclusion

Anti-chimeric fHbp antibodies were able to provide effective killing ofstrains from both fHbp family (A and B). This ability is not altered bymutation of the fH binding site.

Example 14 Immunogenicity of Hap Also Called Map Herein Methods

Antigen Preparations

Map

Native cleaved Map was purified from supernatant obtained afterfermentation of H44/76 cps-strain. Two lots were produced, the secondlot being obtained from H44/76 cps-overexpressing Map [achieved byamplifying the entire map gene from H44/76 by PCR and cloning in aNeisserial replicative plasmid derived from pFP10 (Pagotto et al,Plasmid 43, 24-34, 2000), containing a lacI^(Q) gene and a tandemlac/tac promoter for controlled expression of Map]. Map was purified byconcentration and chromatography steps.

Recombinant Map N-ter (aa 43-1178) was produced by cytoplasmicexpression in E. coli.

fHbp

Recombinant fHbps variants (v) A and B were produced in BL21 (DE3) E.coli after IPTG induction and purification using IMAC column. The fHbpsequences were derived from strains MC58 (fHbp B) and 2996 (fHbp A).Genes were cloned in pET24b plasmid without the nucleotide sequencecorresponding to the leader sequence and with a His-Tag in C-ter.

Animal Procedures

Map

Groups of 10-25 mice were immunized three times by the intramuscular(IM) route on day 0, 14 and 28. Each injection contained 10 μg of nativecleaved Hap or 5 μg of rec N-ter Hap formulated with specol. On day 42,blood samples were taken for serum. Mice sera were from experiments20090608, 20100463, 20100708.

Groups of 6-10 guinea-pigs were immunised three times by theintramuscular (IM) route on day 0, 14 and 28. Each injection contained10 μg of protein formulated with specol. On day 42, blood samples weretaken for serum. Guinea pig sera were from experiments 20090619,20100464, 20100711.

fHbp

Groups of 10-30 mice were immunized three times by the intramuscular(IM) route on day 0, 21 and 28. Each injection contained 5 μg ofmonovalent fHbp vaccine (fHbp A or fHbp B) adsorbed onto Al(OH)₃. On day42, blood samples were taken for serum. Mice sera were from experiments20080790 and 20090265.

Groups of 6-10 guinea pigs were immunized three times by theintramuscular (IM) route on day 0, 14 and 28. Each injection contained10 μg of monovalent fHbp vaccine (fHbp A or fHbp B) adsorbed ontoAl(OH)₃. On day 42, blood samples were taken for serum. Sera were fromexperiments 20090200 g3 or 20100464 g10.

SBA

N. meningitidis strains were cultivated overnight on Petri Dishes at 37°C. +5% CO₂. They were sub-cultured for 4 hours on Petri Dishes with 20μM TPEN (zinc chelator) at 37° C. +5% CO₂. Serum samples (pooled sera)were inactivated for 40 min at 56° C. and then diluted 1/50 inPBS-glucose 0.1% and then twofold diluted in a volume of 25 μl in flatbottom microplates. Then 25 μl of a mix of bacteria, from agar-plateculture or after cell contact (diluted in PBS-glucose 0.1% to yield˜50-250 CFU per well) and baby rabbit complement (final concentration inmicrowell: 12.5% v/v) was added to the serum dilution. After 75 min ofincubation at 37° C. under shaking, 2 layers of agar (0.9%) were addedto the wells. The microplates were incubated overnight at 33° C. +CO₂.The CFU's were counted and the percentage of killing was calculated. TheSBA titer is the dilution giving 50% of killing.

Map KO Strain Construction

N. meningitidis strains growth and transformation procedure wereperformed as described previously (Weynants et al, 2009).

Strain 17540 was a gift from Julio Vasquez (CNM, Madrid, Spain), strainM01-240355 and NZ98/254 from R. Borrow (HPA, Manchester, UK).

The map/hap::kanR plasmid, consisting in a kanamycine resistancecassette inserted into the unique PstI site of the H44/76 hap gene (vanUlsen et al, 2003) was a kind gift of Prof. Tommassen.Kanamycin-resistant colonies were screened for the inactivation of thehap gene by

PCR on boiled bacterial lysate. Integrity of LOS was checked by Tricinegel and Silver staining for all clones to avoid changes in complementsensitivity.

Genetically modified L3, 7 and L2 lipooligosaccharides from Neisseriameningitidis serogroup B confer a broad cross-bactericidal response.Weynants V, Denoël P, Devos N, Janssens D, Feron C, Goraj K, Momin P,Monnom D, Tans C, Vandercammen A, Wauters F, Poolman J T. Infect Immun.2009 May; 77(5):2084-93.

A Neisserial autotransporter NalP modulating the processing of otherautotransporters. van Ulsen P, van Alphen L, ten Hove J, Fransen F, vander Ley P, Tommassen J. Mol Microbiol. 2003 November; 50(3)

Results

Different N. meningitidis strains selected based on their homology forMap were tested in SBA using sera from mice and guinea pigs immunizedwith native cleaved Map or recombinant N-ter Map formulated with specol.As shown by the results (table 1 below), Map induces a cross protectivebactericidal response as strain M06-240336 with the lowest homology withH44/76 is killed. 17/22 strains are killed with anti-native cleaved Mapantibodies obtained from guinea pigs. Six of these 17 strains are notkilled by anti-fHbp antibodies. Difference of expression level of Mapcould be an explanation for absence of killing some strains as suggestedby the facts that strains sharing similar Map sequences are killed(M06-240336) or not killed (M06-240877).

To confirm that Map is the target of bactericidal antibodies, ΔMapstrains (NZ98/254, M05-240355, SP17540) were used in bactericidal assays(table 2 below). In such SBA conditions, no killing was observed. Theseresults confirm Map as a major antigen to induce cross bactericidalantibodies.

TABLE 1 bactericidal titer of anti-Map antibodies in presence of babyrabbit complement H44/ M97- M05- M01- 76 MC58 250687 SP17540 0240072240355 760676 BZ10 SP17662 Map homology (H44/76 = 100%) 100% 99.9% 99.9%98.8% 98.7% 98.7% 98.7% 98.4% 97% fHbp family B B B B B A A A A fHbpexpression ++ ++ ++ − − + − + Immunotype L3 L3 L3 L2 L2 L3 L2 L3 L3 ccST32 ST32 ST32 ST11 ST11 CC213 ST11 ST8 ST269 Animal Treatment Serumbactericidal titers fHbp B sera or A 3539 5786 <100 <100 <100 <100 775256 mouse native cleaved Map lot 1 187 2394 113 <100 769 <100 1578 896lot 2 580 536 5036 510 <100 744 2443 1936 rec N-ter Map 471 1149 23371431 GP native cleaved Map lot 1 781 938 4658 1098 <100 2679 116 61622353 lot 2 4070 4762 12047 5658 143 6800 >12800 11884 rec N-ter Map 82862123 10055 8991 M01- M05- M05- M06- M06- 240101 240210 NZ98/254 0240471240877 241336 DE 10038 Map homology (H44/76 = 100%) 96.9% 96.1% 96% 96%90.6% 90.6% 90.6% fHbp family B B B B B fHbp expression + + + +/− +Immunotype L3 L2 L3 L3 cc ST269 ST41/44 Animal Treatment Serumbactericidal titers fHbp B sera or A 509 <100 388 <100 367 mouse nativecleaved Map lot 1 2742 <100 570 <100 <100 2327 <100 lot 2 <100 1684 <100rec N-ter Map 157 GP native cleaved Map lot 1 4871 <100 1617 <100 <1008561 <100 lot 2 <100 6688 <100 rec N-ter Map 5278 M01- M01- M98- 240013240149 SP18025 DE 10690-06 250771 SP17567 Map homology (H44/76 = 100%)fHbp family A B B B A fHbp expression + + +/− +/− +/− Immunotype L3 L3cc ST269 ST41/44 L3 Animal Treatment Serum bactericidal titers fHbp Bsera or A <100 2104 4373 <100 <100 110 mouse native cleaved Map lot 1lot 2 <100 <100 1217 <100 <100 <100 rec N-ter Map 1365 920 GP nativecleaved Map lot 1 7819 6300 1605 <100 1527 121 lot 2 >12800 >12800 4263<100 6258 587 rec N-ter Map 7254 4448

TABLE 2 bactericidal titer of anti-Map antibodies against Map KO strainsNZ98/254 (fHBP B/+) M05-240355 (fHBP A/+) SP17540 (fHBP B/−) strainsstrains strains Species Treatment WT ΔMap WT ΔMap WT ΔMap mouse nativecleaved Map lot 2 1684 <100  744 <100 510 <100 rec cytoplasmic Map NT NTNT NT 1149 <100 GP native cleaved Map lot 1 1617 <100 2679 <100 1098<100 lot 2 6688 <100 6800 <100 5658 <100 rec cytoplasmic Map lot 2 NT NTNT NT 2123 <100

Conclusion

Map induces cross-bactericidal activity and provide effective killing ofstrains not killed by anti-fHbp including strains from clonal complexST269 (eg M01-240013 strain) and strains from L2 immunotype (eg SP17540strain).

The results suggest that a vaccine based on Map and fHbp will offer abetter strain coverage than a vaccine based on fHbp.

Example 15 Hsf (Also Called Msf Herein) Induces Bactericidal AntibodiesAgainst Wild Type Strains Methods

Antigen Preparations

OMVs

Outer membranes vesicles (OMVs) were produced using classical 0.5% DOCextraction from recombinant H44/76 strain (porA KO, capsule minus, galELOS and over-producing Msf and/or ZnuD protein).

fHbp

Recombinant fHbps variants (v) A and B were produced in BL21 (DE3) E.coli after IPTG induction and purification using IMAC column. The fHbpsequences were derived from strains MC58 (fHbp B) and 2996 (fHbp A).Genes were cloned in pET24b plasmid without the nucleotide sequencecorresponding to the leader sequence and with a His-Tag in C-ter.

mAb

mAb Hsfcross/5 was obtained from fusion of myeloma cells and lymphocyteB obtained from BALB/c mice immunized with 20 μg of OMVs fromrecombinant H44/76 strain (porA KO, capsule minus over-producing Msffrom strain M01-0240101) using a repetitive, multiple site immunizationstrategy designated RIMMS.

Animal Procedures:

OMVs

Groups of 10 guinea-pigs (GP) were immunized three times by theintramuscular (IM) route on day 0, 14 and 28. Each injection containedOMV antigen normalized to 10 μg of protein and formulated with AlPO₄. Onday 42, blood samples were taken for serum. Sera were from experiments20090266.

fHbp

Groups of 6-10 guinea pigs were immunized three times by theintramuscular (IM) route on day 0, 14 and 28. Each injection contained10 μg of monovalent fHbp vaccine (fHbp A or fHbp B) adsorbed ontoAl(OH)₃. On day 42, blood samples were taken for serum. Sera were fromexperiments 20090200 g3 or 20100464 g10.

SBA

N. meningitidis strains were cultivated overnight on Petri Dishes at 37°C. +5% CO₂. They were sub-cultured for 4 hours on Petri Dishes at 37° C.+5% CO₂. Serum samples (pooled sera) were inactivated for 40 min at 56°C. and then diluted 1/50 in PBS-glucose 0.1% and then twofold diluted ina volume of 25 μl in flat bottom microplates. Then 25 μl of a mix ofbacteria, from agar-plate culture (diluted in PBS-glucose 0.1% to yield˜50-250 CFU per well) and baby rabbit complement (final concentration inmicrowell: 12.5% v/v) was added to the serum dilution. After 75 min ofincubation at 37° C. under shaking, 2 layers of agar (0.9%) were addedto the wells. The microplates were incubated overnight at 33° C. +CO₂.The CFU's were counted and the percentage of killing was calculated. TheSBA titer is the dilution giving 50% of killing.

Msf/Hsf KO Strain Construction

N. meningitidis B strain 17567 (from J. Vasquez, CNM, Madrid, Spain)growth and transformation procedure were performed as describedpreviously (Weynants et al, 2009). The msf/hsf::CmR plasmid wasconstructed as followed. Briefly, a DNA fragment of 4771 bpcorresponding to the 1531 bp 5′ flanking region of hsf gene, the 1775 bpof hsf coding sequence and the 1465 bp 3′ flanking region was PCRamplified from H44/76 genomic DNA with primers Hsf sens(CGCAATAAATGGGGTTGTCAATAATTGT) and Hsf reverse(AGTCAAGGCGCACGCTGTCGGCAT) and cloned in pGEMT-Easy vector. The plasmidwas then submitted to circle PCR mutagenesis with primers HSF5′ci2(gaagatctgccgtctgaaacccgtaccgatgcggaaggctata) and HSF3′ci2(gaagatctttcagacggcgataaagtcctgccgcgttgtgtttc) in order to (i) deletehsf gene, (ii) insert uptake sequences and (iii) insert BglIIrestriction sites allowing easy cloning of the antibiotic resistancegene. The CmR gene was amplified from pCMC plasmid (Weynants et al,2009) using primers BAD20 (tcccccgggagatctcactagtattaccctgttatccc) andCAM3′Bgl2 (agatctgccgctaactataacggtcc) primers. This fragment was clonedinto the circle PCR product after BglII restriction resulting in plasmidpRIT15456. Chloramphenicol-resistant colonies were screened for theinactivation of the msf/hsf gene by PCR on boiled bacterial lysate.Absence of Msf expression was further confirmed by Western Blot.Integrity of LOS was also checked by Tricine gel and Silver staining forall clones to avoid changes in complement sensitivity.

Genetically modified L3, 7 and L2 lipooligosaccharides from Neisseriameningitidis serogroup B confer a broad cross-bactericidal response.

Weynants V, Denoël P, Devos N, Janssens D, Feron C, Goraj K, Momin P,Monnom D, Tans C, Vandercammen A, Wauters F, Poolman JT. Infect Immun.2009 May; 77(5):2084-93.

Results

To evaluate the potential of Msf to induce bactericidal antibodiesagainst wild type strains, strain SP17567 with a high level of Msfexpression (western blotting results) was selected.

Sera against Msf or Msf-ZnuD OMVs and control blebs (without antigenoverexpression or only ZnuD overexpression) were tested in serumbactericidal assay. Monoclonal antibody specific for Msf was alsotested.

Results in table 1 below show an increase of at least 20 fold ofbactericidal activity for Msf overexpressing blebs compared to controlblebs. High bactericidal titer is also observed with Msf specific mAb.Only the recombinant fHbpA induces a low bactericidal response aroundthe detection limit of the test.

To confirm that Msf is the major target of bactericidal antibodies, ΔMsfSP17567 strains was used in bactericidal assay. In such SBA conditions,no killing was observed. These results confirm Msf as antigen is able toinduce killing of wild type strain expressing high level of Msf.

TABLE 1 bactericidal titer against strains SP17567 wild type and Msf KOSP17567 WT ΔMsf mAb Msf 2864 <100 OMVs Ctrl OMVs 390 <100 ZnuD OMVs <100<100 Msf OMVs 7821 <100 ZnuD-Msf OMVs 5847 <100 fHbpA 110 <100 fHbpB<100 <100 mAb LOS (L7/12) 896 522

Conclusion

Anti-Msf antibodies could mediate the killing of a wild type strain thatis poorly killed by anti-fHbp antibodies. This suggests that theaddition of Msf to a fHbp based vaccine could enhance the coverage of aMenB vaccine.

Example 16 Effect of Antibodies Against Tdfl (Also Called ZnuD Herein)Overexpressing Blebs on L2 Strains Methods

Antigen Preparations

Outer membranes vesicles (OMVs) were produced using classical 0.5% DOCextraction from recombinant H44/76 strain (porA KO, capsule minus, galELOS and over-producing ZnuD (and Msf) protein).

Animal Procedures:

OMVs

Groups of 26-30 mice were immunized three times by the intramuscular(IM) route on day 0, 21 and 28.

Groups of 10 guinea-pigs (GP) were immunized three times by theintramuscular (IM) route on day 0, 14 and 28.

Each injection contained OMV antigen normalized to 5 (mice) or 10 μg(GP) of protein and formulated with AlPO₄. On day 42, blood samples weretaken for serum. Mice and guinea pig sera were from experiments20090265, 20100463 and 20090266, 20100464 respectively.

fHbp

Groups of 20 mice were immunized three times by the intramuscular (IM)route on day 0, 21 and 35. Each injection contained 5 μg of monovalentfHbp vaccine (fHbp A or fHbp B) adsorbed onto Al(OH)₃. On day 49, bloodsamples were taken for serum. Mice sera were from experiment 20090833.

SBA

N. meningitidis strains were cultivated overnight on Petri Dishes at 37°C. +5% CO₂. They were sub-cultured for 4 hours on Petri Dishes withoutor with 20 μM TPEN (zinc chelator) 37° C. +5% CO₂. Serum samples (pooledsera) were inactivated for 40 min at 56° C. and then diluted 1/50 inPBS-glucose 0.1% and then twofold diluted in a volume of 25 μl in flatbottom microplates. Then 25 μl of a mix of bacteria, from agar-plateculture (diluted in PBS-glucose 0.1% to yield ˜50-250 CFU per well) andbaby rabbit complement (final concentration in microwell: 12.5% v/v) wasadded to the serum dilution. After 75 min of incubation at 37° C. undershaking, 2 layers of agar (0.9%) were added to the wells. Themicroplates were incubated overnight at 33° C. +CO₂. The CFU's werecounted and the percentage of killing was calculated. The SBA titer isthe dilution giving 50% of killing.

ZnuD KO SP17540 Strain Construction

N. meningitidis strains growth and transformation procedure wereperformed as described previously (Weynants et al, 2009). When needed,induction of ZnuD expression was obtained by adding 20 mM TPEN(N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine) in the medium.Strain 17540 was a gift from Julio Vasquez (CNM, Madrid, Spain).

The znuD::kanR plasmid was a kind gift of Prof. Tommassen and isdescribed in Stork et al, 2010. Kanamycin-resistant colonies werescreened for the partial deletion of the znuD gene by PCR on boiledbacterial lysate. ZnuD inactivation was further confirmed by Westernblot on whole cell lysate after growth in presence of TPEN. Integrity ofLOS was checked by Tricine gel and Silver staining for all clones toavoid changes in complement susceptibility.

Genetically modified L3, 7 and L2 lipooligosaccharides from Neisseriameningitidis serogroup B confer a broad cross-bactericidal response.Weynants V, Denoël P, Devos N, Janssens D, Feron C, Goraj K, Momin P,Monnom D, Tans C, Vandercammen A, Wauters F, Poolman J T. Infect Immun.2009 May; 77(5):2084-93.

An outer membrane receptor of Neisseria meningitidis involved in zincacquisition with vaccine potential. Stork M, Bos M P, Jongerius I, deKok N, Schilders I, Weynants V E, Poolman J T, Tommassen J. PLoS Pathog.2010 Jul. 1; 6:e1000969.

Results

Three strains from L2 immunotype and clonal complex ST11 were used inrSBA. These strains are not killed by anti-fHbp antibodies andcomplement (table 1).

The evaluation of the bactericidal potential of ZnuD antibodies wasperformed via the use of zinc-restricted growth media (like in-vivoconditions). This was achieved by using 20 μM of TPEN in MH agar plates.

Mouse anti-ZnuD OMVs sera tested in a bactericidal assay under TPENconditions demonstrated the killing of the three strains tested. Similarresults were observed with sera from guinea-pigs. In the absence of TPENin the culture medium, strains were not killed by anti-ZnuD antibodies.

To confirm that ZnuD is a major target of bactericidal antibodies, aΔZnuD SP17540 strain was used in bactericidal assays with TPEN. In suchSBA condition, no killing was observed. These results demonstrate thatZnuD is the major target of bactericidal antibodies against strainSP17540.

TABLE 1 bactericidal titer of anti-OMVs against L2 strains M05-0240072760676 SP17540 SP17540 ZnuD KO fHbp family B A B fHbp expression − − −Immunotype (inner-core typing) L2 L2 L2 Clonal complex ST11 ST11 ST11Animal Treatment Serum bactericidal titers fHbp B sera or A <100 <100<100 <100 mouse Ctrl OMVs MH-agar <100 MH + TPEN agar <100 ZnuD OMVsMH-agar <100 MH + TPEN agar 3400 1308 2130 <100 ZnuD-Msf OMVs (B2468)(lot1) MH-agar <100 MH + TPEN agar 6590 3182 3433 <100 ZnuD-Msf OMVs(B2468) (lot2) MH-agar <100 MH + TPEN agar 2943 GP Ctrl OMVs MH-agar<100 MH + TPEN agar <100 ZnuD OMVs MH-agar <100 MH + TPEN agar 5311 63158877 <100 ZnuD-Msf OMVs (B2468) (lot1) MH-agar <100 MH + TPEN agar 36312312 9344 <100 ZnuD-Msf OMVs (B2468) (lot2) MH-agar <100 MH + TPEN agar4871

DISCUSSION AND CONCLUSION

It is to note that in previous experiment (see example 3 above), two L2strains (760676 and M05-0240072) strains were not killed by anti-ZnuDantibodies. In repeated experiments (presented in this example), thesetwo strains are killed by anti-ZnuD antibodies. Because the expressionof ZnuD was not systematically checked on cultures done to perform SBA,it is thought that ZnuD was not expressed by the strains 760676 andM05-0240072 in the former series of experiments (presented in example3). One possible explanation for absence of expression was the use oftoo old TPEN plates for an efficient chelation of zinc.

The new results, obtained with WT L2 strains as well as the SP17540 znuDKO strain, confirm that ZnuD (over-expressed in OMVs) provides effectivekilling of strains from L2 immunotype not killed by anti-fHbp. The datasupport the idea that a vaccine based on fHbp and additional antigen(s),like ZnuD, will improve the strain coverage compared to a vaccine basedon fHbp only.

1-36. (canceled)
 37. A method of treatment or prevention of Neisserialinfection or disease comprising administering to an individual in needthereof a protective dose of an immunogenic composition comprising: (a)a first, fHbp, polypeptide antigen and (b) a second antigen compositioncapable of generating an antibody response against a Neisseriameningitidis L2 immunotype.
 38. An immunogenic composition comprising(a) a first, fHbp, polypeptide antigen and (b) a second antigencomposition capable of generating an antibody response against anNeisserial meningitidis L2 immunotype.
 39. The method of claim 38,wherein the infection or disease is caused by L1-L12 immunotypes ofNeisseria meningitidis.
 40. The immunogenic composition of claim 38,wherein the second antigen composition is selected from the group ofantigens consisting of: Neisserial L2 LOS, Tdfl, Hap, Hsf and TdfH, or acombination of two or more of said antigens.
 41. The immunogeniccomposition or of claim 38, wherein one or more of the antigens areexpressed in an outer membrane vesicle.
 42. The immunogenic compositionof claim 38, wherein the composition comprises an additional antigenfrom Neisseria meningitidis.
 43. The immunogenic composition of claim38, wherein the fHbp antigen is selected from the group consisting of:(a) fhBP A, (b) fhBP B, or (c) a chimaeric protein comprising amino acidsequences F1 and F2, wherein F1 is a N-terminal fragment of a first fHbpamino acid sequence; F2 is a C-terminal fragment of a second fHbp aminoacid sequence; wherein said first and second fHbp amino acid sequencesare from different fHbp families, wherein sequence F1 and F2 are both atleast 10 amino acids in length and wherein the chimaeric protein iscapable of eliciting antibodies against both fHbp families; and (d) apolypeptide having at least 95% to (a), (b) or (c).
 44. The immunogeniccomposition of claim 38, wherein the fHbp has at least one mutation toreduce or prevent factor H binding.
 45. The method for manufacture of animmunogenic composition of any preceeding claim, the method comprisingexpression of a first and/or second antigen in an outer membranevesicle, wherein the composition is for use in prevention of Neisserialinfection or disease.
 46. The immunogenic composition of claim 38wherein (a) the first fHbp polypeptide is a subunit polypeptide,suitably isolated and purified to at least 50% purity, and the secondantigen composition is present in an outer membrane vesicle, or (b) thesecond antigen composition is a subunit antigen, suitably isolated andpurified to at least 50% purity, and first fHbp polypeptide antigen ispresent in an outer membrane vesicle.
 47. The immunogenic composition ofclaim 38, wherein one or more antigens is comprised within an outermembrane vesicle, and wherein the one or more antigens has beenupregulated, such that a higher level of antigen is present in outermembrane vesicle compared to the level of protein present in outermembrane vesicles derived from an unmodified N. meningitidis, suitablystrain H44/76.
 48. The immunogenic composition of claim 38, wherein thefirst antigen is combined with an antigen capable of elicitingantibodies against a N. meningitidis ST11 clonal complex strain.
 49. Theimmunogenic composition of claim 48, wherein said antigen capable ofeliciting antibodies against a N. Meningitides ST11 clonal complexstrain is selected from the group consisting of: L2 LOS, Tdfl, Hap, Hsfand TdfH, or combination thereof.
 50. The immunogenic composition ofclaim 38, wherein the first antigen is combined with NadA.
 51. Theimmunogenic composition of claim 38, wherein the first antigen iscombined with Lipo28.
 52. A process for producing an improved vaccinefor prevention of disease or infection caused by N. meningitidisimmunotype L2, comprising the step of formulating together (a) a first,fHbp, polypeptide antigen and (b) a second antigen composition capableof generating an antibody response against an Neisserial meningitidis L2immunotype.